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1 1-1 TO.

II GRAPJ-IY

R. M. ALLEN
SECOND PRINTING

NEW YORK
I),

V I\N N
'250

TRAND
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COMPANY,
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PREFACE
'pid modern advances in the application of the microscope to

hi 'IllS of all sorts, both research and commercial, have naturally reI I III :l simultaneous development of the art of photomicrography.

COPYRIGHT,

1941,

BY

D. VAN NOSTRAND COMPANY, I NC.


All Rights Reserved
This book or any parts thereof, l1WY not be reproduced in any farm 1vit/UJut 'written 1Jcrrnission from the p'llbli8h~rs.

First Published October 1941


Reprinted April I943

PRINTED

IN

'rITE

TlNl'1'ED T'HID

,'I'A'I'E8

01'

AMlDHlCA

BY THE PUMP'I'O

1;:1. NOltWOOD

. MASS.

I" In can no longer be classed as a hobby, indulged in only by a 11 rollP of amateur microscopists, it has become an essential adI II) Ihe use of the instrument itself in practically every scientific I unuucrcial field. pill' of this, authoritative reference books on the subject of Cl 11 11 l'I"ography are lacking. Whether this is the cause or the effect 1111 11 non idea that expert knowledge is unnecessary for taking piehrough the microscope is immaterial. The fact remains that micrographs (of a sort) are being taken daily by hundreds, if I nusands, of microscopists, in every line of microscopic endeavor, sole qualification for the task is a fundamental knowledge that 1111.1 re formed by a microscope can be projected onto a sensitive to ruphic film and be reproduced thereby, IloII better pictures can be taken as one's experience and knowledge Ih' technique increase is to be assumed, as is also the fact that the lCl\'ir. of individuals can learn more rapidly and easily by assimilaI CIf rhe published experiences of others than by the slower process rsonal plodding. This is the reason for the existence of this book. It hough the close relationship between visual work with the miII l'OPC and photomicrography would seem to have warranted inhi iun of thc latter in the author's book, The Microscope, this was II f -nsihlc because of the large amount of material which would have I 110 he included to cover the subject in a thorough manner. The It runt ivc was the complete segregation of the two subjects and their publication separately. wor I dealing primarily with the photographic aspects of microsnp\' cannot, of course, undertake an exhaustive discussion of the ele11 lit :11')'optics of the microscope to the same degree as a book pri1I111111y Oil the instrument itself. Some knowledge on the part of the 1111 'I'nsl'npisr must be assumed. On the other hand, it is desirable that I \\ III I, oil phot omi .rography be sufficiently self-contained to be unI I tood without constant rcfcrcn .c to outside matter. This implies

VI

that some duplication of subject matter COil 11 11011 to both visual and photographic work must occur - so much, at least, as lIlay have a bearing on the production of ideal photornicrographs. The material contained in the present volume is derived almost exclusively from the author's personal experience, which has extended into practically every known application of the microscope, and is passed on in the hope that it will prove for many a substantial short cut to a working knowledge of photomicrography, For the benefit of those already familiar with the use of the microscope, the text has been so arranged that only matters of direct interest need be considered, without wading through irrelevant matter, On the other hand, beginners can follow through each chapter in the sequence presented, and after becoming acquainted with the subject from the standpoint of basic principles and the various ways these have ?een worked o~t in mechanical design, can take up the practical technique of photomicrography. The chapter on photographic processes has been added to provide information on this phase of the work for those who may not have had previous experience with them, I wish to express my appreciation of the wholehearted cooperation extended to me by various companies in providing cuts of the various apparatus and equipment shown, These include, The Bausch & Lomb Optical Co. Spencer Lens Co. E, Leirz, Inc. Carl Zeiss, Inc. Eastman Kodak Co. Radio Corporation of America, Cooke, Troughton & Simms of York, England, and others. R.M.A. June 1941

11 I 1111 I1 11 11 I \I PIUNCII'II:S OF PIIOTOl'"IICIlOGIlAI'IIY , 1),1111111"11 HI'I,II ion uf 1-,hot01l1i'l'Ogr<lphy to ordinary photography CIIIIql111 '"1 III 1Ilil'l'os('OPl' to n camera lens lnfinity distance Focus of I," H,'I.IIIOII of sin' of image ro size of object - Effect of the focal 10 1I}1111 ,d .1II'~ls Oil lilt, r~l;llivt: illlage size ,Spherical and chromatic abcrI "1"" \\'III\(' light Sp .cuum Refraction - Correction of lenses for 111111.111"11 (:irl'k IIf confusion EfT t of lens aperture on the size of Ill, .111111'111111 NUIlI 'ri 'al apcrture-I':1l1pty maffnification - SY111~11et111" .11111 1I1111('t rint! lenses Depth of focus -1 he compound rmcroI "I" lis pi'ill('iplc of operation and constituent pans:- The optical sys11111 lit Ill!' l'IlIlIlHllIlllI micros 'ope - Objectives - Eyelleces - Condensers 1111' I II!'IIi('Iit'o I :1SPCl'IS of resolution - Fffect,of ~he size of, the ,clrcl~ of IlIlItll 11111 1111 F"I'IIIUia f'or resolution - IIlU1111llatlOn - Critical illurnina11011 I llll ('1'1'111 met hods of securing critical illumination - Requirements 10 I" 1111'1 III .,('('mil1g .rit ical resolution in photomicrography,

""'1

10111 H

\'1111111 III IUlr:llAl'llIC EQUIP\1F. T ,

1111I ('IIII,t! \I:II'IS t'olllpl'ising a photomicrographic outfit - Various types I1t 111111111('11'1.1 (''l"ipI11l'11\ available to supply the wide range in demand1111111 ,III.II'II('d ('lIl1ll'l'a'> Their advantages and limitations - Simple verIh iI I .111,,'1,1'>l Iorizouml-vcrticnl outfits Large research models - Self11111101111('11 unt I'r,al oulfils Photomicrographic apparatus to meet special I '1"111'1"('111\ lquipmcnt for l<,lw-power phoromacrography - IlluminaI hilI I 11'"1"11('111 1,;l1I1P~ of varrous kinds - Accessory eqUIpment - Fea11111,\001I,IIIk 111 IIlinos 'opcs to be used III photo1l1lcrography, 110 11 1\111 l'I)III'i\IENT, III 1,111011 of microscope and .arncrn in makeshift apparatus - Conditions III I" I I11 1'>ldl' 1'('" ill 111(' design of horncmadc apparatus for photographyI \ I" of ("",,('1'.1 desired Suggcst .d basic designs in wood and metal con11 lit 111111 tll1' llOl'i'lo1l\;tI, vertical and combination outfits - Methods of 1I1111111'Iilll1 i\I01l1l1il1gS for various pieces of apparatus-Making a 111111 1.1h(,llo\\s C;OI111(Ttionbctwc n the camera and microscope - ReI 1111111' (01111'01 of Ih(' intensity of the light source with ,high intensity 1'lIlIp C:olldl'I1Sillll. Il'l1Sl'S r .quircd Means for colltrolllllg d~e expo1111' '1','('Lt! hOIIl('lImdl' illuminaring system, for metallurgical and "1"1'1"1' \\ urk SlIl'P(l1'ting tnblc Ior i hc I'hOWllllcrographlc outfit, 1111 1IIIINII)"1. IHI PllIll'OMIC11001lA1'IIY SI

107

1'1 11111111111'\' ('ol1.,idl'l'llI iOllS The WOl'i<n)OIlI Conditions to he 111'1in it 111111"1'11,,' 11/1JlIII'lIt"s Opt'l'llIiollS in Illini(,:II~IS in pll~l\Ol1li~'r(),Wllplly .11 "11111' Olllil'll nlif.tIIIlII'11I !l.liMnllll'Ilt, of \,l'I't1('n,I,o,III(II~ I I!SIlI~)11 of 111111111 I'IPlilllillnl'1 I1'Slill!( of Ill(' l'lJ"II'I1I('~1I ('I'ltl('1I1 II1U!"111:1t1011 11111 .IIHI fdll" fol' JI"olol"it'I'oWlll'hv 1\1I,,'('I1III1('(1(IS ('lJIIIJlIIIl'lll I'C

v ii

VIII

II'NI,

CIfAPTlm
I'

cm

quircd for. thc ph(!tolllitT()gr~phi<: tuhl . I{n'ol't! (':\I"(I~Iur l"POMII'C d:lI:\ - The optlca~ cqurprncnc desired for:1 'Olllpil'H' r:tllgl' of Illagllifinniol1s Effect of various factors on magnification Till' practical determination of magnlficatI~)J1 - Filters and their characteristics raking the picturcFactors effectIng: the nrne of exposure. - Dctcrmining thc basic exposure :- The computation of exposures - Miscellaneous considerations _ Choos~ng the proper filter - Pictorial composition in photomicrography _ MeetlI1g .unusual conditions in the object - Curvature of the field - Optical sectJOnl11g- Securing del?th of focu~ - Inherent limiting conditions in photomlcrogra~hy - SectIonal. map pIctures =: Use of very high magnifications - ~upeIlmpos1l1g eyepiece scales - Lo~-power photomicrography - Tra~smmed and opaque --: Common faults 111 photornicrographs - The author s method of critical illumination by Imaging the light source. 5
SPECIAL PHOTOMICROGRAPHIC PROCESSES

Chapter

11' TAL PRI

I PLL'~ OF PHOTOMICROGRAPHY

IllIIflUIII

l\IIetallography - Dark field photomicrography - Polariz~d ligh~ -'Ph~to~ mlcro~Taphy m narrow spectral bands - Ultra-violet - Infra-red - MotiOn-pIcture photomicrography - Photomicrography in color - Various color pr?cesses adaptable - Stereoscopic photomicrographs - The elec, trorn c mIcroscope. 6.
MICllOPHOTOGllAPHY . . . . . .

h \ ol'll/lf.,ofO'lllicrog1'Clph is a compound of photograph, a picture


of light, and micrograph, an object. Originally" micro11Il':1111 only a drawing, made by free hand, or by means of a I 110 rnph, liS these were the only methods known for producing the I III d illl:1g'c. The application of photographic processes of reproI1 IICIII 10 t hc mi 'J'oscope provided a new method of securing en1 I pictures of minute objects, hence the combined word phototJ III/,h. This compound word necessarily is limited to mean a I a 1\l'II through a microscope, by means of light acting on a I 111 i cmulsion, but the word micrograpb used alone has had to be 0111 ncd in Jl1c:1I1ingto include pictures of minute objects either I 11hv hand or produced through photographic processes. I nmn cr of fact, photomicrography is so rapidly replacing hand 111S (made by means of a camera lucida attached to a microI ) Ih.u it is probably only a question of time until the term micro-: ,/I \\ ill come to mean 0171y a photomicrograph. norhcr Icrm introduced to cover a particular class of photoIII raphs is photomacrograph (incorporating the Greek word for 'IIIa/..'ros), meaning a photographic image of a relatively large I, 'I JII:1g'ni(ietlonly a few times, i.e., not over ten diameters. A till 'I'ograph " is, then, a slightly enlarged picture of an object. uruct illlcs Ihe word microphotograph is used for photographs I 11wit h Ihe microscope, but this word is used incorrectly, for it 11\ ":r minute photograph," which must be examined as a micro'PI' oh jl'(.'I, 1Irider thc microscope, in order to observe the details of pi IIII'l'. The relation of microphotographs to the microscope, and III "hod of producing them, will be considered in Chapter 6.
1111 III III \I I 11h

d throu rh rh

instrumentality

d rcprodu .rion of a minute

11

Defined - ~isto~y o~ - Uses of - Method of takin~ - The proper of lenses for this work - Plates for - Setup and details of procedure. 7
PHOTOGRAPHIC PROCESSES, MATERIALS, A 'D EQUIPMENT. . . . .

type
. .

Chemistry of d~velopment and fixation -.The darkroom - Equipment and ap~ar~tus reqUlred.- Glass~are -:-Ch~mlcals - Developing ~nd printing technique - Reduction and intensification - Making lantern slides - Formulas of developers and miscellaneous solutions. 8.
ILLUSTRATIVE PHOTO MICROGRAPHS . . . . . . . . . . . . .

300

A selec?on of fifty photomicrographs with the exposure data for each, illustrating various fields in w:h,ch photomicrography plays a part - Showmg low and 11Igh rnagnificarions, various techniques, etc.
INDEX.

353

I," ."

..

Id

11\

11"1\11'11111.I11'11

ClI'I'IIClICI,IIIII()I.II\l'ln

1111 \IIIIN

Ill.

1'111111)

11I1I1I1.1(\l'ln

III

IIll1lIN\R\

PIIOTOGRAPIIY

Relation of J'hot(}'IIIicrogrll/J/.ly

to Ordillllry

J'f.,otogr1pby

The possibility of taking pi .turcs by means of i hc micros 'ope is dependent upon the fact that basicall y there is no di IT crcncc between a camera lens and a microscope. They both obey the same laws of optics in producing an image of a properly illuminated object. As a matter of fact, photomicrography and ordinary photography merge so perfectly into each other that it is difficult to say just where one leaves off and the other begins. The best place, therefore, to commence a discussion of the optics of photomicrography is with the camera lens. In every case, a camera lens must be of.svhat is known as the" positive " lens type, i.e., one that is thicker in the center than at the periphery. Only a positive lens (the term of course includes compound lenses functioning together as a single positive lens) can produce a real image such as is required for photographic purposes. Negative lenses are those which are thinner through the center than at the periphery. These, or combinations of lenses which have a resultant effect similar to a negative lens, cannot produce a photographic image. The image in this case is known as a virtual image. When an object is located a great distance from a camera lens, ordinarily known as " infinity," the rays from the object are brought together by the lens to form an image of the object in what is called the focal plane of the lens; the distance of this plane from the optical center of the lens is .designated the focus * of the lens. For all practical purposes, the distance" infinity" can be considered as anything over 200 times the focal length. The size of the image bears a definite relation to that of the object, which is the ratio of the distances between the lens and the object, and the lens and the image. Thus the image of an object located 200 times the lens focus from a camera will be r/20o actual size. Only when the object is located at infinity will the image plane be distant from the lens the exact focal length of the latter. As the object comes nearer, the image plane moves farther away from the lens, and the ratio between the object size and image size changes accordingly. This, of course, is why cameras must be focussed for the proper object distance, if one is to obtain sharp pictures.
,. Technically, this is known as the principal focus. Actually, every lens has two foci, one on each side of the lens. These arc called conj/lgate foci. The .onjugarc focal point corresponding to the principal focus is located at infinity. s it COIlICS nearer the lens, the corresponding conjugate focus recedes.

'hen Ihe objc('1 is bro\lglll I();\ certain dist~lI1cefrom. d:e lens it is rluu the illlage plan' has rel'et~ed to a pom~ w~1ere It ~sthe same I mcc [rom t he lens as the object distance. ~h1S dlst.a~ce ISfo~nd to act Iy t wi .c the focal. leng.th. UI~der th~s c,ondltl.on, the 1mag.e I identical with the object SIze. WIth ordma~y hand cameras tlus IlIwt be accomplished, because not enol:gh adJl1s~ment or. be~lows I Ih is provided; therefore, if phot?graphmg an object full SIze ISdeI. a sI' .cial long-bellows camera ISnecessary.
IIld

ubi.!t

I.~ t~
I(
Object

OD)

.
1(.

",i:f,~n<
ID-'1

01 1I\I{"ily

of

Object at twice focal distanc

Object between focal point and twice focal distance Image nmgnijied I' I. Relationship

between

Image size and Object

size.

(1: 0 :: ID : OD)

th . bellows is sufficiently long, it is not obligatory to stop wh~n 11 IZ' has been reached; bringing the object ~till nearer results 111
image plane farther away and the Image then becomes Thus the ?ame ca~11eralens b~I in 'ff en a photomicrographic len~ producl~~ possibly a mag~l1non of s .v Tal diameters, These vanous conditions are shown dianuunt icnlly in Figure r. . 111 [ur we have not taken into account the effect of a change m o ill lcn rth of the lens on image formation. H~re the parallel ben 11 c.uucra lens and a microscope becomes still .closer .. Let us 1 I f 'W examples and apply the simple mathematical ratio to the I IIld illlagc size. 1111' all ohjccr 10 inchc high located roo feet distant to be
III I he

t r Ihall the object, or magnified.

I"JNI>" II'NI \1. I'IIINCIJlI.I', 01' 1'II0101\11IltCIC,II \I'II\,

1111. \ liON 01' 1'11010,1IIII0C,II \1'11\ 10 OltlllN 1\\ 1'1I0I'OC:I(AJlIIY


I

.5

photographed by a .arucrn equipp 'd with ;J () inrl: ( [r.) IeIlS. Its photographic image is ro" X r/200 Ilw of ;111inch high. Using a I2-inch (I ft.) lens under the same conditions, the illlage size would be IOn X 1/100 = 1/10 inch. In other words, when the distance is fixed, the size of the image can be increased substantially in proportion to the increase in the focal length of the lens employed. This will hold more or less true as the distance of the object from

FIG. 2.

Effect of the focal length of the lens on image size

the camera is decreased. But with extreme nearness of the object other factors enter the problem. We have already seen that any lens is ~apable of producing a full-sized image when the object is distant twice the focal length of the lens from the latter. With a 12-inch lens this occurs when the object is 24 inches away. It also requires a camera bellows length of 24 inches, while with a 6-inch lens the object yields a full-sized image with an object distance of I foot and only a I2-inch bellows length is required. Should we wish to make a several times enlargement of the object we would discover that a r z-inch lens is almost prohibitive because of the excessive bellows length required for the camera; the 6-inch lens is far superior in this respect. AS5U1l1-

Ihlll 0111'111:1 imum hl'lIows 1l'1Irt h is limited to 36 inches, it is cvithat l'llIplo~'illg a still shorter focus lens, say a 3-inch, would hi a much I{ighcr IlIagllilic:1t ion to be obtained, Going to a i, "liS would provide a possible Illagnification around 70 diameters. I h:ls 1\0 hearillg at ,Ill ()n.th~ fllndal1l~ntal.Iaw;'~t only I~eans that I IlIld we wish to secure a 5111111ar l11ag11lficatlOn WIth a I a-inch lens, vould I' 'qtllre a bellows in the form of a tunnel about 7 5 fee~ long. nmlit ions arise both in ordinary photography and photomicrogIln where lenses of longer focus are required to meet certain c~nnns, These conditions, as will be pointed out later, ha.ve to do WIth I 'lat ive area of the object 'which must be included I? the photo1 h; t he longer-focus lens accommodates a correspon~mtsly greater I. Figure 2 illustrates graphically the effects of van~tlOn .111 the " kllgth of the lens on the image size ~nd ,the w~y m which the I of t he object included in the field of VIew IS restricted by the use horrcr focus lenses. question might naturally arise in.connection 'with tl;e statement udv made that a i-inch lens used WIth a bellows extension of 3 fe~t " vicld a magnification of some 70 diameters: ," Why not use a still "I' bellows and obtain magnifications of 160 or even s~veral h~nI diameters, with the same lens?" The answer to this questIOn ides the justification for introducing the micro.scope ar:d ~icro11 - lcnscs into the picture. It might appear sufficient to dIsmISS the -stion of magnification by means of a long bellows who!ly on the I of its mechanical impracticability, bnt this is not the pnmary obion to it, In the first place, the i-inch lens proposed is already. out of the .class ordinary camera lenses and can proper~y be consIdered. a n:IcroI . lens. There is, in this respect, a conSIderable. overlap'pmg ~n the ton of short-focus camera lenses (even excluding motIOn-pIcture mcras) and long-focus photomicrographic lenses. The fOfl1-:er exI I down to less than 2 inches and the latter up to at least 4 inches lOo 111111.). There is, however, as will be demonstrated later, usually ulicul difference in the design of the lenses for the two purposes. In the second place, a more obvious solution would be t~ use the III bellows and reduce the focal length of the lens. A t-mch lens l on :1 36-inch bellows would double the magnification obtainable tli i1 -in .h I ns. This is the basis of the actual procedure employed. the produ .rion of hig:hly magnified photornicrographs. Mere.ly 1 01'\ cllillg' t he focal length of the lens, however, would not be sans'1

1'1 11\ 11 1\1 1'111 C'II'II CII 1'1111111 III IICII,II \1'11\ factory; there are c .nuin optical luxvs iuhcrcu: ill tilt, problem of producing magnified illl:lgCSof all objc .( ill which SIruct urul details are to be revealed in direct proportion to the .unount of cnlurgcmcnt secured. An understanding of these laws and the manner in which they are mer in the design of microscope lenses is essential to a thorough knowledge of photomicrographic technique. But before they can be taken up a discussion of some fundamental optics is in order. Spherical and Chromatic Aberrations in Lenses from a single piece of photographic or photo-

1'1111t1C \1 \ 11CIIIICII \IIC \1I11t1(\IIONS I 1.1'1\1'1,:1'

Unfortunately a positive lens constructed glass is not satisfactory for either high-quality

White Li{Jht

c --------------~----~

I I Plane of Violet

Plane of Red Inuure

Imaae
FIG. 3.

Spherical

and

chromatic

aberrations

micrographic work. This is because certain aberrations are present which interfere with the production of a perfect image. The ideal condition is represented in Figure 3A, where all the rays proceeding from a point source of light located at infinity are brought together to form a point image. But what actually happens is that the rays passing through the outer zones of the lens are refracted excessively so that they come to a focus nearer the lens than those through the central zone. This effect, shown in Figure 3R, results

, IIt, f:lCI Ihut lens surfact's urc ground as segments of spheres. It 1 tI \ 11as .\/)/.J('fica/ ahcrmtion .. When the image of a point is no I I1 poillt hili :1 fairly large circular area, the image of a concrete 1 \\ hich should he conceived of as composed of an infinite numI OiIlIS,cannot possibly be sharp but will be fuzzy in proportion I 11111111111 of spherical aberration present. I 11 ,I raill; white light is not a single entity but is composed of rays 1111 :11 different frequencies, or, in other words, possessing dif11 \ IIVl' lengths. These different wave lengths affect the eye difh ; t he sensation experienced is what we know as color. The 1il's of wave lengths affecting the eye, and which are largely ihlc for the production of a photograph, is known as the specIt l' tends from the shortest wave, the violet (about .4 micron 1 tit) through indigo, blue, green, yellow, orange, and red 11 ,7 micron wave length). The spectrum is familiar to all from 111111011 occurrence in nature as the rainbow. I 11white light, containing these various wave lengths, is passed It 11lens and refracted, t it is found that each different color is .nl~I1",1 diifcrcntly, those at the violet end are bent considerably h III Ihose at the red end. This fact explains the rainbow, which I hy sunlight passing through raindrops, each of which funcI minute spherical lens, thus refracting the color differently. I mu ion of the various wave lengths, due to the difference in I 11 11It Ihey are refracted, is known as chromatic aberration. It It d ill Figure 3C, . 11I11 -hrornatic light (i.e., one particular wave length only) ntplovcd for photographing, chromatic aberration would not \ it h securing a sharp image, but as the focal length of the lens III for each wave length, it must be focussed for the particular 1 d. Because the focal length of the lens varies with the wave Ih . lII:1gnitude of each color image is, of course, different, so I Il Ihey are all superimposed in one exposure, the resultant P"Ol", 1111 It 'Iy, optical glass of great diversity in both refractive index I
h
11111"1"011 is 1/1000 of H millimcter, roughly about r/25000". dl'lh'l'liCln or bending of light rays occurring when they pass from one III 1111111 lur, such as frolll air into glass (or the reverse), is called reiraction. h IIr 1I1t".I\lIrlllll1ll of refraction is known as the index of refraction, air being .111I111\' (I,on); the rcfracti c index of ordinary glass runs around 1.5. The III I hI' l'(,rl',I(" ive index of glass for different wave lengths of light is its

Ft

\I'll' "1)\ 11<I \I 1'11i"'I.lI'U. 01' 1'11010lie UOe,l!


I

I'I"'IIW\!.

;\

\I

1.11111I I Ill. ,\111-1111:\1111 IN


(I~

II'N

I_

I)

and dispersion is available and through, l',Olllhin,al iom of posil ive and negative lenses made from these glasses, It IS posslhl ' ~() prod lice a COll,lpound photographic lens, whethe~ of call1e~a or IIl1crosc<,>pe typ~, "'. which both spherical and chromatic aberrations are practically c1U11Inated. In such lenses, rays of light of all col?rs pass tI:rough, all ~ones from center to periphery to meet at substantially a POInt, as In FIgure Even assuming perfect compensatIon for b?th sphencal and chromatic aberrations, the vibratory nature of lIght Itself prevents the formation of an absolute point image of a theoretical point source of light, The image inherently possesses a positiv~ ci~cular area, T!1e condition is illustrated in an exaggerated manner In FIgure 4, The CIrcular disc image of the theoretical point is ~alled the circle ,Of confusion, * It is aggravated in size when spherical or ch~o,mat1c aberr~tions are less perfectly corrected, but the determining factor In establishing the theoretical minimum diameter for any given lens sysAnti-point Point Source in Object FIG, 4, (Circle of Confusion)

P 'I 10 Ih~' I' .lnrivc vulue lal'~' apcrturcd lens's 10 <.::III1CI':I III I ph()IOIIlIl'l'0W:lJ~h)' I~l're,ls tillS fundalllental ditf .rcnce. In of ,Ihl' form 'I' 11 IS 1)1'1111:11'I1 of advantage to secure greater IIh -nn r power, usual referred to as the" speed of the lens,"

3A,

"

_~~~~~~.
. ""-'

Small Apcrtl,ro
Laroo Anti-point

~__ -===~~

Laroe A1Jerture

Small Anti-point

IhTI'l'nSt' in size of the anti-point with increase in the aperture of the lens

The image of a point, formed by a lens, is. never a 'point, but a cir.cular disc of definite diameter, known as the antl-pomt, or CIrcle of confusion

tern is the wave length of the light employed, The dia~eter of the disc is reduced as the wave lenzth decreases. Or, stated In another way, sharper pictures can be secured with the same lens by using blue light than are obtainable by using red, From a practical standpoint, for any g~ven wave length, wI:en enlarged images are involved, the greatest SIngle factor m reducmg the size of the circle of confusion is the angular aperture of the lens, In Figure 5, two lenses are shown diagrammatically which are ass1.~med, to be alike in every respect except that the angular apertu~e of B ISt~lce that of A, Or, to state it more accurately from a technical standpoint, the trigonometric sine of one-half the total ~n~le of th~ limiting rays enterinz B is twice that of A, Then the antIpomt, or CIrcle of confusion, otthe former is one-half as large as that of A and the ability to resolve detail is accordingly twice as great.
. From the microscopist's standpoint the" circle of confusion" is often called the

" Airy disc" or antipoint.

pressed in terms of the ratio of the effective diameter of the fot'~1 length, the term.f+ 5 implying, a focus for infinity Cl +s tunes the largest diaphragm opeOlng, With the dia11 on the same lens dosed to one-half the diameter (i.e, set at I light would be diminished to one-fourth the intensity, and 1 nrly an exposure four times as long would be required, \ IItolllicrography, the object is usually stationary and the length pllsure is therefore immaterial. What we are interested in is more structural details, Indeed, in proportion to the extent of I nncnt secured, this is the only reason for wanting a magniI' in the first place, I the use of a greater aperture in microscope lenses is only inI 11far as the light-gathering power is concerned (although of 11\ high magnifications, as otherwise unduly long exposures he necessary); the prime importance lies in the increased ability I' ,':lt,l'r detail: . -I he I,nost c?l1venient way ,of expressing this 1'!SIIC of n nucros OplC lens IStherefore not 111 terms of f ratio, IlIlIcri{'~1 /l pcrt ure. This term was originated by Erncsr Abbe s t hc sine of Oil .-hal! the angular aperture, multiplied by the
, Its

10

I'll

11\

"N I \I

I'IIINI

11'11'.

01'

1'11010

11111111,11 \1'11\

1'111'1111 \I

11 111110

I "le:

1I1'1I11"ION~

IN

IINSI'S

11

refractive index of t he medium operate. It is written

wit h which t hl' km is dcsi rued to


1],

. A. =

sine

111'1 a'klls must hl' Wowd ill t he \nl~' it was lksigncd to be used Ill, froll! of thl' Il'IISIII1IS! always fucc toward the longer of the \I ,foci, The shorter cOlljllgate focus is always that next to the

a result of this,

11

Ilsy III11ICtrical lenses designed for distant

in which .A. is numerical aperture, 12 is the refractive index of th medium, and U is one-half the angular aperture. With all lenses designed to work in air, 1], is equal to unity and hence actually plays no part in the equation. The limit of enlargement of which any lens is capable can be demonstrated mathematically, basing the figures upon the size of the circle of confusion. Beyond this limit further increase in image size results only in empty magnification, i.e., magnification without corresponding increase in resolution of structural details. This limit is usually expressed as roughly lOOO times the N.A., for the optical center of white light (around .55 micron). Thus a camera lens 'K' which has an N.A. (equivalent1of f:4.5) of .11 would have a theoretical limit of magnification of I IOX irrespective of its focal length. As such magnification is a low power from a microscopical standpoint, one limitation upon obtaining highly magnified iinages by the expedient of indefinite increase in bellows length is evident. But there is still another very important factor to be taken into account, which affects the application of ordinary camera lenses, no matter how highly corrected they may be, to photomicrographic purposes. As already pointed out, corrections for spherical and chromatic aberration involve the use of compound lenses, often made up of several components. Figures 6, 7, and 8 illustrate some of the well-known camera lenses on the Tessar (unsymmetrical) market. One characteristic common to two of these lenses (the Goertz FIG. 6 Dagor excepted) is that they are not symmetrical when viewed from the front and back. In ordinary photography there is an additional condition to be satisfied, known as astigmatism. It has been found that to overcome this, as well as spherical and chromatic aberrations, and at the same time provide fast lenses at a reasonable cost, unsymmetrical combinations offer one of the best solutions. Hence many of the well-known camera lenses are of the nonsymmetrical type. But to obtain the best results an unsymmetri But corrected for the object to lie in the plane of the shorter conjugate focus.

<

Dagor (symmetrical)
FIG. 7
I

11hy are not even idealfor copying at full size and produce an
IIllage when the longer conjugate focus is at the back of the n syrnmetrical lens, such as the Dagor, Figure 7, it is obvious 1'1 he front or back could be toward the longer or shorter con'us without affecting the operation. Hence, if camera lenses I employed for taking low-power photornicrographs, one I take sure they are of the symmetrical type, of which there rl on the market. As an alternative, it often suffices to reuusyrnmetrical Iens for enlarging purposes, so that the light object enters the back - i.e., the front of the lens is turned to-

(ilm.
I I .rurcrs

of long-focus

lenses intended

especially for photo-

11hy compute them for this purp~se, so that naturally they 11 superior to camera lenses of equivalent focus and aper~ure. fundamental principle, 'well known to all camera enthusiasts,
s with large apertures possess correspondingly less depth of 'I -cinlly with nearby ob. f onc focusses on a per"..-_+-: 1 :t few feet from the vcrything in front 'and our of focus. When ~ :> d 'pt h of focus is desired,
III 10 "

stopping .down " losing t hc diaphrngm Ul11 ircd cll'cct IS obtained.


\'Ot'ds,

II' .rturc.

we work with a This principle

Sigrnar (unsyrnmetrical)
FlG.8

12

l'lINIlA,II<NI

I. I'IIINUI'I,I',

(11' 1'11(11(1, IIi IUU,II

I'll'

1111, ((I,

II'(II'NI)

11111(1 (01'1',

11

applies to photomicrography as well: :111 illl'l'CaSl' ill Ihl' "rduces the depth of focus, although to the h 'lIdil (If grl':lIl'r resolution. In low-power photomicrographic lenses use is nuulc of :Ill iris diaphragm for this purpose, just as with camera lenses, but with objectives of higher power the same end must be reached by the substitution of' another lens of lower .A. As a result of this possibility of securing either an approximation of an optical plane or an appreciable depth of focus, at will, the trained photomicrographer possesses a means of meeting a wide range of conditions often present in microscopical problems. " The Compound Microscope

I' I 1111 he rcmov 'd ill will and (IIll' of different magnifying 11 IIIUll'd, Pmcticull ull umnufacrurcrs now make their reg0,""11 of '\ l'/lil'l'l'S I(I Ihe sa Ine SInnd.ird diameter (23 mm.). Also, 1/11 .11 (Ill t hc bottom or the tube, known as the Royal So-

b"I'I'III(J(ltialo l nnuio

Thus far, in establishing a relationship between photomicrography and ordinary photography we have assumed merely the employment of suitably corrected lenses of diminished focal length and increased aperture, for securing high-power photomicrographs. In theory this is possible, even to the highest power, but in practice it becomes necessary to obtain extremely short-focus lens equivalents by compound magnification through a microscope. Low powers require single-lens magnification only, and for this range it is not even necessary to employ a microscope. Lenses for this purpose generally start at about 100 mm. (4"); then a complete series is available, such as 75 mm. (3"),50 mm. (2"),35 mm, (d"), 20 mm. (t"), and I 6 mm. (-V'). At this point we reach the realm of the compound microscope. As the name implies', the compound microscope effects its ultimate magnification by remagnifying an already magnified image. The lens nearest the object, designated the objective, functions just as would a camera lens arranged in the manner described for producing an enlarged image of an object. This enlarged image in turn becomes the object on wbich the second lens, called the eyepiece, or ocular, is focussed. The image formed by the second lens is the one projected onto the sensitive film to produce the photomicrograph. This is shown diagrammatically in Figure 9. As the compound microscope is an essential component of all highpower photomicrographic equiprnents, we will pause for a brief study of its mechanical design, a simple model of which is shown in Figure 10 with the various parts designated. The objective and eyepiece are mounted on the ends of the body tube, the former by screwing in place, the latter merely by a sliding fit.

'l'hi become the ill111" ;''''Lod'object of fit" H(I('ond stane

0/ "",unifr,caLion Projected image


I'll., Cl,

Schematic diagram of the compound

microscope

Id. h:ls hccn adopted by all manufacturers for both the rnituhc and objectives, Hence not only all objectives and eye11 I, b)' :lny on company are interchangeable, but they can be I 11 'd with those of other manufacturers as well." I spect, however, complete interchangeability is limited by !tzur ion of different tube lengths for the microscope. Some I 1\ ' adopted 160 mm. as the distance between the shoulders hi -h the objective and eyepiece rest, known as the mechaniI Il rh, In other makes 170 mm. is standard, while microI lid 'd for metallurgical purposes have tube lengths ranging to I 5 111111. This variation in tube length limits interchangerh ' optical parts due to the fact that objectives are computed I If, 'I results only when used with a definite tube length. For III it is always necessary to make sure that the tube length of o 'ope is correct for the particular lens employed. This can h 1011' hy an adjustment of the draw tube into which the eye-: , ,und which in turn slides into the main part of the body tube. ubi' 'live and eyepiece, together with the body tube, constitute I I 111'(' 11 [cw exceptions to this general statement;' as in the case of specia!

lip "

where a larger-diameter

eyepiece is employed,' and low-power

photoof older

phlt It'ilSt's of large aperture which of necessity are larger in' diameter than
111111111111:111'11

11Ill'

(olll1d

hy the Royal Society thread, Also, many microscopes tluu employ larger-diameter eyepieces.

14

I'll I)A,\II' I \I, 1'1(11.11'11' 01' 1'1I01t1.III,IICII.II.II'1I

1111 (Cl

II'CII'11

IIC110C01'1'

-----

OCULAR TUDE

RACK----~ PINION ofcoarse Rdjuslme~L----

---

BODY TUBE

__

REVOLVING NOSE PIECE

'-

FINE ADJUSTMENT

OBJECTIVES

/ --

CONDENSER STAGE

r
____

SUBSTAGE

I_FILTER GLASS CA.RRIER

SUPPORTS

11527

I
CLAW FOOl' BAS.Ij:

11

Cross section of Apochromnte Objective FIG. 10. Cross section of a compound microscope (Zeiss) with the various parts named. At the lower left is shown an apochromatic objective in partial cross section. Although ten separate lenses are required in its construction, they all function as a single lens.

1'"111111~cd Ih(' IlIil'l'IIS('OP(' Ilhidl [uuct ion like the photographic 10 IllIdlll'('lfll' ('III:ll'gnl illl:l 1(', " f 11':111('11 q h of rill' IlIil'l'os('o(l" object ivc be so chosen that the 011 h"1 \\,('('11 1I1l' cOlljllg:1ll' foci of the object and image planes is /11, Ih,; iuuurc formed by the objective is magnified 10 times, We " 11d,'sigllal 'Slll'h ohjc .t ive as a IOX,implying that it possesses an ,111I.lllIilw:llioll of-r o diameters. I IIll1POlllld microscope the position of the image plane is always I h\ Ih(' mnnufact urcr so that it will lie at the proper place for the " III pick it lip and further magnify it. But as the latter can h(' [ocusscd, in combination with the objective, to yield a sharp 1111 t lu- sensitive film, no matter where the latter may be located, 1I11('S Ill'l'CSS,lI'Yto csta b lish a reference distance corresponding to 1111 uilir.u ion one obtains when using the microscope for visual I " This distance is the ame as that assumed as the distance of I ion for rhe normal eye, i.e., 10 inches. If, then, a screen is I III inches from the eyepiece (specifically, the Ramsden circle, poinr, where the rays cross, just outside the eyepiece), the image I d Oil it will correspond in magnification to that seen by the eye I" work with the microscope. This means that if the eyepiece . 11" a I Cl times enlarged image formed by the objective would be 11111 rn i fi cd 10 times, making 10 X TO or IOOX total magnification. III .1microscope capable of yielding a I OOX image at a r o-inch dis1111 he conceived of as the optical equivalent of a single lens of , [ocus. onsidered in this manner, the relationship between \I photography and photomicrography, whatever might be the " ';11ion of the microscope lenses employed, is easily recognized. lnuccar the microscope depicted in Figure 10, however, reveals h 'rt' arc other parts to the instrument, in addition to those re110 form the image. From the optical standpoint the principal , these is rhe condenser, located beneath the stage. Its function is 1I to illuminate the object on the stage when the latter is being d or plHlrographed by means of tra~1sparent illumination. Clll" r('spe'r photomicrography is radically different from orphotography. In rhe latter we are dealing almost exclusively' IIlrj"CIS which arc opaque and therefore photographed by the aid hi reflected fro IIIrhem. Only occasionally ate silhouette photoIa""I1, wit h Ihe- source of illumination located behind the obI h,' condition IIslI:1lly cricountercd in photomicrography is the Ill' t Iiis. I [ere I h ma jor part of the work is done with light

16

FUNI)AME'TAL

PRI

'CII'LJo:S

OF

PIIOTO

IICllOWtAPIIY

! Ill

01' III

\1

11', I

01'

1111'

co,

11'(l\"

11

IIU(O~<:()I'II:

17

passed through the object, the latter being sufficiently thin and transparent to enable this to be done. Only in the photographing of surfaces of opaque objects is the condition similar to that of ordinary. photography. The employment of transmitted light necessitates the addition of the condenser, whose function, as will be discussed in detail later, is to condense or focus an intense beam of light onto the object. The fact that the three optical p'arts of a microscope require proper alignment with each other, rigid support, and means ... (both coarse and fine) for adjusting the focus to secure a sharp image on the image plane, explains the need for the other mechanical parts of the instrument. Also, the object must have a stage upon which it can be located with respect to the objective. Further, it usually happens in visual work, and often in photomicrographic work, that the source of light is located somewhere away from the optic axis, so that to pick it up and project it into the condenser a mirror is required. , This, in a brief way, gives the reasons for the essential parts of a compound microscope. The actual design of the instrument may vary over wide limits, but basically in everyone will be found all the features necessary for general work. Higher-priced outfits have many additional features. These may include circular rotating stages, mechanical stages for moving the object (i.e., the glass slide upon which the latter is mounted), more complete substage apparatus with decentering diaphragm, binocular body tubes for visual work, etc. The Optical System of the Compound Microscope

(a) Objectives The most critical piece of optical equipment of the compound microscope is unquestionably the objective. Especially is this so in photomicrography, where the demands made upon it are even more severe than with visual work. Imperfections in the image formed by the objective, due to the latter's falling short of perfection, are naturally aggravated by further magnification with the eyepiece. Moreover, the initial magnification of an objective intended for high-power work may amount to well over IOOX, though it is unusual to employ eyepieces higher than lax to 2OX. Whatever the magnification of the eyepiece it cannot possibly bring out any resolution of detail not already present in the image produced by the objective. All it can do is to make such detail as is present more easily seen by the eye.

III 'obj :ti c is rh 'I' iforc of prime importance to III 'opisl alld ever uuinufacrurcr of microscopes endeavors to IIIllUl'l' 1h ' IIIIIIOSIpossible. As rcgards the degree to which aberraI '.lI('l'n eliminated, objectives can all be grouped into three I hcsc arc designated Achromats, Fluorites (or sometimes I I -hnnuurs), and Apochrornats. I 1(1111,11 ic oh jecrivcs are corrected for spherical aberration for 1111(dcsignurcd the preferred color) of the spectrum, usually 110v rccn, as this is near the central portion of the visible band. I luul imperfections due to spherical aberrations in the regions h Hit, of the yellow green are thus minimized, although they rcurcr in proportion as the color is removed from the pre0101", . 1111 ion 10 spherical aberration correction for one color, achroIll" ' -rives are also corrected for chromatic aberration by superI I \\'0 spectral col or regions so that they also come to a focus III 'point. Doing this brings all the other calor regions close tond a Cl' good image results, especially for visual purposes. I 11I1I1:Hic objectives are still more perfectly corrected. This is through the use of the mineral fluorite, which possesses char.rlltl,,'H not obtainable in any known glass. Spherical aberration is I for two regions in the spectrum instead of one only, as in I \I i ' objectives, and three colors have been superimposed in cor11'.hromatic aberration, thus making such lenses almost ideal. lit uion only is present in apochromatic lenses: they require 'OITected eyepieces known as compensating oculars to be unbinarion with them, because a part of the correction is acI Iin the eyepiece, . I ird S .rics of objectives, known as Fluorites or Serni-apochrolilt 'l'lll xliarc between the other two, being much superior to Ii ' I 'nscs hut not quite the equal of apochrornats. Like the I require compensating eyepieces in order to assure their

I 'I f '('lioll of

o muncc.

I 11,11 work, high-quality achromatic objectives are very satiss a matter of fact, only an expert microscopist would be. 1'1 'ct the difference in the images produced by achromatic hronmric .obj.ectives, unless they were seen simultaneously uid of a comparison eyepiece. * . As to photomicrography
ClIIII'"ri Oil (. '('pi('et is a 11 ,1111'illlll!((" Irmn IhcIII I I 011' hulf of Ill(' lirld is hi i 111111Ihl'Olllfh Ill(' device which can be fitted over two microscopes, being brought together into one eye lens in such the view through one microscope while the other other mi roscopc. .

18

Ftl

1>1'1 110.Ii'll. I'IU~LlI'I.I<S 01' 1'11010IIU\()(,HI'II'II\'

1111Ill'IIC\1

'11

I Ill' 1111ell, 11'111'110011 111,110, (01'1'.

11)

'j
11
1

with achromatic objectives, however, their performance differs markedly from that of apochrornats. Only when employing :1 green filter which passes the particular spectral region for which the lens lISually is best corrected does an achromat yield an image approximating' the results obtainable with an apochrornat. On the other hand, for many purposes a fluorite objective is practically the equal of an apochromat, at least in the center of the field. (b) Eyepieces Eyepieces ordinarily consist of more than one lens component, usually two, the lower of which is known as the field lens, the top one being designated the eye lens. They can be divided into two general groups - Ramsden, or positive combinations, and Huygenian, or negative combinations. The latter are far more commonly used than the positive type. It may seem a contradiction of the statement previously made that a negative lens is not capable of yielding a real image, that a negative combination can be used as an eyepiece to magnify further the image formed by the objective. There is, however, actually no contradiction involved. It will be found that a Ramsden, or positive, eyepiece can be used as a hand magnifier, while a Huygenian cannot. On the other hand, a negative lens can take an image already formed by a positive lens and modify its size. In Huygenian eyepieces, both components are positive elements; it is only in the way in which they are related to each other that .the combined effect is that of a negative lens. Actually, in the formation of the ultimate microscopical image, either visual or photographic, the two components function separately to produce the desired result. A more important classification of eyepieces from a practical working standpoint is the division into ordinary and compensating eyepieces. Both of these can be of either the Huygenian or Ramsden type, although the former is far more common in ordinary and compensating oculars, the Ramsden is limited largely to the high powers of the compensating type. The primary purpose of compensating oculars is to effect a.final correction in the degree of magnification of the blue and red Images of apochromatic objectives, which are not of the same size. Use of compensating oculars obviates this defect so that the images coincide on the photographic plate. . . An objective is capable of producing a magnified Image on a photo-

II (, " when lI~t'd wirluuu :111 t' cpiccc, ndcr this condition III t' .trt lY as if it we n' :1shun Iorus cnrncra lens, with the ob"' . It'sS thun the imn rt' distancc. The llIagnification is, of , III 'h less I hall where an eyepiece is employed, being equal to 11
I

' . - X initial magnification I It'l1glI1 0 f 0 I>Jectlvc I I 11It'

[rum

ohjl't'tive

to

plate

,.

"

of object

IIllUC'lIHl'd :11101 her way, the distance from the optical center of the 10 the pial " divided by the equivalent focus of the lens, asthe imu re is formed sufficiently distant to be considered at
11 is COl1lll1011 practice among some microscopists to utilize hod of ohraining lower magnifications than they can secure 1/ Iivc nnd eyepiece combinations at their disposal. Results 111 I his 1I1:1I1ner arc never ideal unless achromatic objectives, I for infinity, arc employed with a bellows extension approxiIIIll11il v, or both arc corrected for the same finite distance. objectives of all kinds, intended to be used with eyepieces, I 'd for a definite tube length. They perform properly 'only I1 xmdirion. When the tube length is extended many times hich the objective is corrected, the resulting image is infe-

I I
11

I Inion to this condition apochromats are subject to a further


11,Ihut of a di fference in the size of the blue and red images, to Icrcncc has already been made. This can be taken care of, I hut horh the blue and red images are required to register on , only through the use of properly corrected eyepieces. Of I ould color filters be employed to suppress completely all 10'" ha nd of color, this difference in the size of the calor I 0 longcr a factor. 'lIhstagc Condenser 111 Ion us ' of the microscope with transmitted light necessi,loving a condensing lens or system, located beneath the stage, I III \)OSC of con 'entratin~ a ~one of light on the object, como I I' apcrture of the objective, . 11 microscopes for visual work are equipped with but one I, \\ hich rnusr therefore be more or less universal in its adapt11 IIIl' entire range of objectives. When used with an oil11\ uhjccrivc having an aperture of .. 1.30, to secure. the
11

20

DA'I\II':

TAL

PHI

CIPU:S

OF

JlIIO'J'O,\IICRO(;JtAJlIIY

Iln

1111'OI\l<IJC\L

ASJlH.IS

01.

ItFSOI.U'f'IO,

2I

utmost result of which the lens is capable, the condenser must also possess a possible aperture of I,30 .A. To provide for this variation, an iris diaphragm becomes essential, and is therefore always furnished as "apart of the substage. Condensers are available in simple, uncorrected types, known as Abbe a-lens and 3-Jens condensers. The latter gives a maximum aperture of N.A. I,40, while the former is limited to about .A. I, 20. In either case the top of the condenser must be in oiled contact with the bottom of the slide in order to secure the maximum .A. If not oiled, the numerical aperture of any condenser is limited to .A. 1.00. Reducing the size of the diaphragm opening effects a reduction in the aperture of the condenser to adjust it to the desired N.A. In addition to uncorrected condensers, others are available, in which spherical or chromatic aberrations (or both) are corrected, just as in objectives. For photomicrogTaphic work it is highly desirable that a condenser corrected at least for spherical aberrations be available. Such are known as Aplanatic condensers. The reason for this will become apparent as we discuss the theory of illumination pertaining to transparent obj ects. " . In addition to the ordinary high-apertured substage condenser, a complete outfit for photomicrography in the low magnification ranges must include low-power simple lens condensers, as well; the extremely short focus of the standard condenser is capable of illuminating but a minute portion of the area photographed at low magnification. The Theoretical Aspects of Resolution

1IIllllhillg 10. Iht illlagc formed hy the objective; it can only en11 Ihal tkl :111 a Ircady present can be seen by the eye. In photoI I~lh\' si ill another fa .ror contributes to the ultimate enlaroe 0 I liS is IIll' bellows Icngth. Increased magnification secured by IIf .1 Ion rcr bellows is similar in effect to the employment of a "\\ er eyepiece. It cannot succeed in bringing out detail that is 1I IIlIa"Tt'[onucd by the objective. 0

F,G. I I. Engraving on Glass, IOOOX III.l(.(lIific:lliol1 resulting from use of an objective

of only .30 N.A.

Having considered the design and purpose of the optical parts of the microscope, we are now in a position to examine some of the factors related to the formation of the best image of which any given combination is theoretically capable. As already pointed out, there is a limit to the resolution obtainable with any objective and while there is no actual limit to the amount of magnification possible, all beyond a certain limiting value gives no increase in what can actually be seen. After all, the only purpose in producing a magnified image of a minute object is to reveal additional information as to its structure. When we reach the point where enlargement provides no additional revelation of structure, further amplification becomes worthless. We have also seen that the ultimate possibilities in the way of resolution are controlled, or rather limited, by the objective. The eyepiece

I I und 12 illustrate the difference between empty magnifi, IT,(,tin' magnification. The former is a photomicrograph I 1I1:II:gt'1I1CIlt has been pushed far beyond the point where I III ':11 inn ends. The object is one which lends itself adIhi P:II:I icular demonstration. It is a line engraving done 11I11I111011 glass, the work of Mr. Alfred McEwen of Tarry.""" nor sharp and clear and the writing cannot be read,
I

Ihllll IIf IIl11ldllf.[Ihest: minute writings and the machine used to produce lib 11III 1111 111'1 il'k IIy Mr. Mcliwen in the Scientific American for June, \ I hlllf.t undoulit xlly represent the finest handiwork of man. The, hll\ll1 1tI'I'I.I'III1I:liIlS the Lord's Prayer written in a space only 35 11 IIIIt 11111'. Ol1ly 10 those accustomed to thinking in terms of minute r th 1 IIf.tlln:s 1111':/11 !llIylhillg', It convcys a better idea to the lay mind 1111 III 111".'1 il h \I hich we arc familiar. In one squa,re inch this same I h 1I111'1111I11'd 1I1',ld, I7fJ,()()(} times! This means that the entire Bible, I 1 II 1011111'111\. l'Ol/ld Ill' written at the same size within the area of I "'1.,1/ I" ttuu () 'c,,.i ' .

22

1'\)1'<1)\\11',1\1. 1'111111'11 (11' 1'11(11(1,1111\(11,11\1'11'


It)

Ill'

1111(1111111 \1 \ I'll I (11, III 011110

although the magnificalioll of loo~,.di:II,I1l'll'rS shouh! hI.' .uuplc. II i~ obvious that increasin~ thc. ll1aglldlca,II.(1I1 10 d()~~hk I h.u employed would not help in deciphering the wrttlllg. III I"glln.: r z we sec th ' . same object at a mag~ificatiOl: O,f 1500X. l Icrc the cnlargelllcnt has not been secured by hIgh eyepIecmg and long bellows length alone but
f -

1111,1 '(' (If :I ItIlIHllolI~ /loinl, ;lIld t hc rl'l:lIiollShip of the 1[1 11111('Id ;111 Oh)l',CII\'l' 10 IIll' sil',(.' of this .irclc of confuIII lid\' been 11I1.'l1llOl1l"d. II has also been stated that the 1111111,1 rJli/lC;lIioll has been set at approximately 1000 times bl I'It'.' tllIl'l'rlll~'l' I"f the system. \Vc are now ready to examine \ 111I'11 tillS flgllre hns been determined,

11
B

cl

c
size of circle of confusion

D
on resolution

"Ht'I'1 of

FIG, 12.

Taken with an oil-immersion objective of A. The fine lines produced by the .diamond point are invisible as .w~:itten, They have been made visible by filling them Il1 With a soluble, dye, Hence It is th~ dye retal.nee! in the lines (and some still on the surface) that is actually seen, TI1lS ,results 1~1a somewhat smeary effect, although the ?etail is so perf~ct that, on the original pnnt, the grain of the dye can be easily seen 111some of the lines.
1500X, 1.05

Engraving

on Glass,

by the use of an obje~ti:re cal?able ~f yielding more detail as well as higher magnification m Its pnmary Image. . From this it is evident that there are certain fundamental laws relating to the formation of ideal images, with which ~he photomicrographer should be familiar. Inasmuch, as the eyepIe~e, and b.ello:"s length enter the problem only as .amplIfiers of th~ original objective image, it is clear that we can consider tl~em as an integral p.art of t.he entire magnifying unit, and deal onl~ m ter~s ?f the ultimate SIze of the final projected image for any glVen objective. The presence of a " circle of confusion" in place of an absolute

III Ihl' moment ignore any part the human eye may play I I Olll,\' the effects present in the image as formed by a lens. , ~.I '.I't havt"'two lines ~f a width n separated by the dish' l' lines can be conceived of as a continuous series of It unct cr 11., Therefore, when we photograph these lines, , I III IIll'II,1 will b~ repr~duced, not as having a width n but ollfllSIOI~ havll1g a diameter of as in Figure I 3B, and of the lines would appear in the photograph as in the If 11. Should the diameter of 121be sufficient for the circles I in Iigllre '3C, the photograph would no lonzer show the . purure narrow lines, but as one broad line as in the lower " TI1l1s as dimension d is progressively diminished, it is 11 10 reveal the lines as separate, the size of the circle of cont h 'l'llIlIl' dccreasingly smaller, or, in other words, the aperI ns t'"lpl()~cd 1.11LlSt be proportionately increased. I IIf confusion IS formed as a result of diffraction, i.e., the If li rlu ~vhich ~)ccUJ.. s when light rays pass through an aper,llIl'I'lIlg vanes WIth the wave length of the light (as well 11111' npcrturc}, and hence this f~ctor ~1USt ente~ into an hI 'h expresses the amount of qIffractlon, the SIze of the II1fllSioll; or; what is more practical from the microscopist's I h ' cl ' rrcc of resolution of which any lens is capable.

n"

FU

Ill\\ 11> 'I, 1/\1 PIU (11'11'''' 01, 1'IltllO lit IttU.1l \1'11\

1111 1IIIoIU III \1 \ IJoC.I~ 01' IU':-OI.\)'JION

The trigonometrical formulat ion of I he resul 11 I ion charucrcrist it's of a lens in practical terms is somewhat involved, and is not of direct interest in microscopical work, but the final formula as derived is rcla . tively simple and should be memorized and understood from the standpoint of its effects in the production of ideal micrographs, It is:

R=--.2

A ,A.

in which R is resolution, in terms of the minimum=distance (in microns) between two points in an object which will juSt be revealed as separate points in the image. A is the wave length (in microns) and N.A. the numerical aperture of the system. To find the number of lines per inch which will be resolved, the value in microns derived from this formula is divided into 25 AOo. It is apparent that the question of magnification does not enter this formula at all. This is because magnification must take into account an entirely different condition - that is, making the final image sufficiently large to allow the resolution of which an objective is capable, to be seen by the human eye. Just as actual detail exists in the object itself which is much too fine to be seen by the unaided eye, so can it exist in a magnified image of the object which.is still not sufficiently large to be seen. Thus in a photomicrograph it becomes the function of the eyepiece and bellows length further to enlarge the original objective image to a point where the eye can appreciate it at the distance of best normal vision, i.e., 10 inches from the eye. . The fact that the function of tbe eye plays a part in the final magnified image - whether tbe visual image observed on looking into the eyepiece, or the photographic image recorded on sensitized filmsmeans that we must approach this part of the problem from the standpoint of the eye before we can associate resolution with magnifica-

"'""1111'111 vuuld hl' fOllnd 10 \,:11'\' . wirh dilfcrcnr individuals' , somc III I 1\\0 sl'par:lll' lines much cioscr than others can. With III tI , \\ hen till' separal ion reaches 1/100 of an inch, they I I I , l'I'y [cw (';111detect lines separated only r/20o of an I I I 10 iurhcs [rom Ihe eyc, I t is important to retain the 1111 111011 as ;1 I'd .rcn 'C standard, since the movement of '0 I 1'0illl in .hcs from the cye would be equivalent tlIlI1IW Ihl' all rlt, whi .h in turn is as if the lines were moved I Ip,lrl. Only very near-sighted individuals can focus 11ohjl'cl ~ inches away, and so this is not a normal cons, the 1/100 inch (250 microns) separation use in-determining the resolution and Oil l\l'l'eSS:lr.v under working conditions. Though we 11 h ;1 considcrarion of the separation of two lines, 'it must 111I h:ll Ihe SHIl1C condition obtains in the case of two sepa"Id Ill'IICl.' we can talk in terms of either lines or points , (Ill ils illlagc) indiscriminately, . have an object, such as a diatom, which has line markI 11 ,1I'l: Sll:ll.'Cd 1/ I 0,000 of an inch (2'5 microns) apart. I 11111 I 11 )C magnified in order that the lines in the image I h\ the eyc and the structure of the object itself be reIII d .IS a fair value-to

Ill';d pllrpos

I "'I of the computation is easy; the 1/10,000" must be 1111 il iI :1ppea rs 1/ I00", which would be 100 times. Any IIIIClIIIlt would not be sufficient to allow the eye to discern .1111 s.
I
I
IIIIISI

already sccll'tha.t.mere magnification is not enough; have the ability to resolve the structure at this
by reference to the resolu-

non.
In Figure I 3D are two lines, separated a distance of approximately one inch. With the eye placed 10 inches from the lines the sine of angle which they subtend will be I/ro. The angle corresponding to . r 0 is 5 i degrees. The eye observes the lines with this spacing as very widely separated. But if by some means we could move the lines closer together we would reach a point where the space between them would be just visible; any further movement 'would cause them to blend into one apparent line." Thc distance between them under * Since the eyc Icns obeys the S~I1lC 1~\Vs as other lens S we must cxpcct diffraction
to be prcscnt, and thus a circle of confusion exists in Ihe illlag in the eye, just as in

011. This must be determined

R 2

.A:

\,1', however, the circle of confusion is not the limiting factor. We i11t' mill of :1 sensitive film, or thc screen upon which an image ~s 1111 IIIIII,"I~ ('Olllillll()US in area, but this- is not the case-in the eye. The 11 lip III Ilk n-t inn 'by rI.IC rods and cones, and hence is analogous to the ,j hI 1111 1'1('('11'111 ypl.' pnntlllg. 1n order to be observed as separate lines, rh 11111'III 11ll' ('~'l' IIllISI fall upon different rows of cones in the fovea. I /I Ill' Cl ICIHl'I'hl'l', regardless of thc circle of confusion present, III 1111'1IIIl's filII ClIl Ihe S:III1C I' .tinal cones, the eyc can no longer
fir

I"

l'!"'('

26

FUNDAMENTAL

PRINCIPLES

OF PHOTOMICROGRAPHY

THE THEORETICAL

ASPECTS OF. RESOLUTION

which we will transpose to N.A. A can be assumed to be a green light with a value of .500, which is substantially the approximate center of whit~ light. R will be 250/100 microns if the magnification is set at IOOX and the allowable circle of confusion is 250 microns (i.e., 1/100 inch). Inserting these values in the formula we have: The required N.A.
= .500

2 X 250/roo

or N.A.

.r o.

This numerical aperture is r / r 000 of the magnification, and hence it is common practice to state that the limit of useful magnification with ordinary white light (central at approximately .5 microns wave length) is rooo times the .A. of the objective. Assuining the highest possible correction in the objective and all other factors approximating ideal conditions, photomicrographs taken at a magnification of rooo times the numerical aperture of the objective will be wonderfully crisp and sharp in detail. They should stand examination under an ordinary reading glass without appearing to lose sharpness. This suggests that often, in actual practice, the magnification can be pushed considerably beyond the theoretical limit, with even an apparent gain in detail. The reason for this is usually overlooked. It lies in the nature of the image as a whole which is seen by the eye. The total angle of vision of the eye is very large but acuity of vision is limited to one particular minute spot in the eye, known as the fovea. When we wish to examine anything critically it must be focussed exactly on the fovea. Surrounding the fovea is a larger area, the macula, which provides less perfect vision although still of a high order. Beyond this the quality of the image drops off considerably. As one reads the letters on a printed page, it is done by a process of scanning, each letter in turn being passed rapidly over the fovea. But it is much easier to read large print than small because the imperfect image of the larger type can be partially interpreted by the retina before reaching the fovea, and the concentration required becomes materially less. Examination of a photograph follows the same law. The eye must scan every portion of it (subconsciously, of course) to pick up all th detail revealed. Therefore, if we enlarge the image until a large portion of its interpretation can be effected with the area of rhc retina lv

ond the fovea, fatigue is materially lessened.and the pleasurable non which results therefrom is heightened. In other words, we t he picture with less exertion. At the sam~ time, concentration particular detail will reveal that there is less sharpness than at 1Ill1l'ge1l191twhere theoretical "empty magnification" is not I I. l-xamples of magnifications far in excess of rooo times the . 11':d aperture of the objective employed will be given later. I n, for any reason, it becomes desirable to check the magnificaI umcrical aperture, wave length, and desired resolution against 11 h 'I' in connection with the resultant pbotomicroO'rapb, it is lit t hat the resolution formula need only be transposed to put 101' sought on one side 'Of the equation and all the other factors ClI her side. we must break down R into two 2N.A. factors, the diameter of the circle of confusion ~esignated iIh ' llIagnification CM). As long as we are satisfied with a circle 11 ion of 1/ roo inch (250 microns), d would always be 2.50; but ihlc under some conditions that greater sharpness might be I rh en d might be any value between 250 and a low limit of uns, the latter covering the extreme resolution of which the , is capable. R in the formula will then be replaced by h ' following are the possible arrangements of the formula, I unicrical values derived on the basis of the problem already husic formula R when tile resolution is required,

= .: _A_,

lI

d lE

=
.2

5. X .10

= 2.5M(250)

100

A__ when the m;merical aperture is required; X diM. .


2

,1\.

-X 250/roo

5_._

= .roN.A. is '

X.I f.

..\.

when the diameter ~f the circle of confusion


I'

'quin.d.

FU

DAME 'TAL PR IN CIPLES OF PHOTOMICROGRAPHY

CRITICAL ILLUMINATION

I.e., (4) M

5 X
2

100 _ .10

11 thod
2S0}J-

could be designated. This term is critical illumination: Just hat is critical illumination and how is it related to image formation?

2N.A. X d w h en t h e magm ificati .' d . cation ISreqUlre

'ritical Illumination
/
'

t..

i.e., M = ------

.10

250

100

diameters

5 (5)

t..
i.e.,

2N.A. X d when the wave 1 . reqUlre . d. engt h IS

'

t.. = ------

.10

250

100

S}J-

The ordinary substage condenser is, in effect, a very short-focus 1 ns combination, quite similar: to an objective, but facing in the opI nsitc direction. Rays from a distant light source are thus brought to focus by the condenser, to form an image of the light source. The Oil denser position is then so' fixed (by adjustable focussing means) hilt the image plane of the light lies directly upon the plane of the ob-r, upon which the objective is focussed. This condition is shown' Figure 14. The maximum angle of the cone of light forming the =r+>:
Plane of" Object ~

Thus it is evident that by memorizing the simple basic formula for resolution, one can easily ascertain any factor necessary to meet a given condition. Illumination The important role played 'by illumination .in t~e production of ideal photomicrographs of transparent preparations IS largely Ignored by the average microscopist. This, rather than ~he quality of the optical system, is mainly responsible for the mediocre results so often achieved. Even the best of objectives can perform but poorly when the illumination is not correct. This is equivalent to saying that the performance of t~e objective can be modified or limited by some other part of the optical system, which is true. In the formation of the image the objective can utilize only rays which reach it fr~m the object, an? hence i~ su~h r.ays are not sufficient to produce an Image of the quality the objective IScapable of forming, the image will fall short of ideal by the amount of the deficiency. It is the function of the condenser to provide adequate and ideal illumination of the object. This has nothing to do with the intensity of the light; it concerns only the angular relation of the cone of light formed by the condenser, to the aperture of the objective. Early recognition of this important relationship between the objective, condenser, and illumination source, on the part of some of our pioneer microscopists, resulted in th~ :vorking out of the l~lcthod of producing th b sr results ,111<1 th C011ltngof :1rcnu hy which the

--~--COndefLser Ap."tltre to eqltal Objective N.A.

\ \

, ,

--7---

~
Substage Condenser

c.;

Aperture of Condenser

Princi~le of critical il!umination, where the image of the light source is formed In the plane of the object .

1 nscr image is shown by the outer dotted lines, assuming the I hrugm to be wide open. By closing the diaphragm to the 'point
the r~sultant cone. of light from the condenser is just equal to III ' \~Iu~h c~n be picked up by the objective, theoretically perlIul1llll~tl.(m 1~ provided. It is to this' particular arrangement that I 1111' critical illumination (or critical light) is given. I unl Y II ndcr this condition that the full resolution characteristics uhj .crivc can be realized. The part played by the condenser is t d. i'~ rI.1C resol,utjon fo.rmula which we have already considered. 11III Its fullest form It should actually be written:

'A
(ohjectlve N.A. ~ condenser

.A'T

FUNDA 1EKTAL PRINCIPLES

OF PHOTOMICROGRAPHY

CRITICAL ILLUMINATION

3I

This is because the effective numerical aperture system as a whole is the average of that of the objective and condenser. When the best. possible condition obtains, the N.A. of the condenser is equal to that of the objective and the formula reduces to: .

R=

A
2N.A.

The poorest condition is represented by no condenser at all. For instance, taking a concrete example, where an objective with an .A. of .50 is used without a condenser (i.e., the N.A. of the latter issub-' stantially 0). Then we have which becomes: R

ruplc expedient of focussing the image of the luminant directly, withIt I he interposition of a luminant condenser. The condition was as llusrrated in Figure IS A. In the early days of microscopy, suitable I hr sources, such as we have now, were not available, and so diffir!tit,s were present both in the securing of a light that would cover I entire field and also in obtaining an even illumination in such porillS of the field as were covered. Figure- IS B illustrates what the Ip .urance would be on looking through the microscope, with a
Substage Condenser.,.,

=~
5
lA ht
SOU'1'ce

"\ A

2'-----j<-

111

Image of Flame

In other words, without a condenser the resolution is just one-half or

I,

It might appear from this that increased resolution would ~esult from employing a higher aperture in the condenser than that available in the objective, but this is not possible, because of the introduction of a new complication. This will become evident when we discuss darkfield illumination. The N.A. of the condenser must never exceed that of the objective. . The theoretical aspect of critical illumination is quite involved. There are two schools of thought, each of which attempts to explain the underlying principles of resolution on different premises. It is not necessary that the practical microscopist be familiar with the fine points of the controversy in order to do the best work. It is, however, desirable to be able to appreciate the two conditions which obtain, under slightly varying arrangements of the light condensing systems, now supplied with photomicrographic equipments. Both are of value under certain conditions, so that a knowledge of when to employ one method and when the other is decidedly worth while. To make the situation clear, it will simplify matters to approach the explanation from the historical side. As originally conceived, criri 'al illumination was obtained by rh'

I'll;. 15. The most primitive

conception

of critical illumination

I I

IS I he Iigh t source, under the condition of critical light. The r qucntly used artificial light source was the flame of a-kerosene 11111 .d edgewise, not radically different in general aspect from a'

nrc several ways to obtain a greater coverage of the field. mpl 'st is to move the light closer to the microscope, but because 11' "Ilely short focus of the Abbe condenser even this could r Ihe image of the light source to cover the entire field when ""11\\.1['1' objectives were in use. tI 'I substitute. was soon found in the employment of a so-called , condenser," located in front of the light source a distance IIt ' focal 1t'llgth ofthe condenser: The rays would emerge 'olldl'IlSl'r parallel and would thus represent the condition It I.", rcr source of illumination, reaching the substage conhou h cOlllillg from infinity, as shown in Figure 16A. The

JDAMENTAL PRINCIPLES

OF PHOTOMICROGRAPHY

CRITICAL lLLUMl

ATIO

33

focus (t) of the substage condenser would be a little shorter than i-; in Figure 15 A, under this condition; hence the condenser must be moved a little closer to the object to form an image of the light source, but when properly focussed, the image would be materially increased in size, as in Figure I 6B. The weakness in this method of securing critical illumination lay in the unevenness of the lighting over the entire field. This is ineviPlane of Object Substage '" Condenser",

r rhan the position represented by its focal length, until the rays 1111 it, instead of being parallel, were slightly converging, coming to 11 'liS on the diaphragm of the substage condenser. The path of under this condition is as shown in Figure I7.' With the substage .1 I .nscr in the position to provide critical light, it is found that the 111'1eye condenser has become tbe apparent light source; the flame is III1Igcr seen. This method of obtaining critical light was known 11 1I11:t~ingthe light condenser" in the plane of the object. It was I OI'irinally conceived of as being any different in principle from I Id illumination,obtained by imaging the light source with parallel
Principal
feWIlI; of

"'1

Light Condenser

: ..---- -"'

'\ ~f
"

A
Flame

Image of

COr/den8er

Substage Diap/L7'agm

~.
I'uht
IIIII"'fl

~l

Substage CondenSe)' \ ~

(7)'

"'Light Condenser

Imaoe of Light Condenser

B
As seen in the
Microscope

('"il icnl illumination secured by. imaging the light source in the plane substage dlap)lragm. Sometimes called Kohler illumination

of the

FIG. 16. Improvement

in critical illumination secured positioned to yield parallel

by employing rays

a light

condenser

101\1a bull's-eye condenser. Or, to put it in another way, the 'condenser, when imaged in the plane of the object, was conI unulogous to a disc of ground or opal glass, situated in the III It101.1 as the co?den~er and illuminated evenly all over by the Ill' , , 111 back of It; this ground glass provided both greater area I 'VCIldistribution of the light than was possible with the light

table with such lighting, as each portion of the field is illuminated by the rays from a corresponding point in the light source. Whatever variations might be present in different parts of the flame would be evident in the image of that flame used to illuminate the field of view. To obviate this condition it was often suggested that the position of the substage condenser be altered to throw the flame image slightly out of focus. But immediately this is done, the lighting is no longer critical. With amateur microscopists continually striving to secure uniform illumination over the entire field of view by juggling the relative positions of the substage and bull's-eye condensers, it was inevitable that a slight change in the position of the latter would be found to work wonders in providing an evenly illuminated field of view. This chang cons~sted in moving the bull's-cyc condenser a little nearer the micro-

ilouc.

I1I ill effect this is substantially (although not altogether') true, III d for A. Kohler >J!. to demonstrate mathematically that illuI oht:lill~d in this manner is theoretically different in principle I " I' 'slIltlng from imaging the light source itself in the.plane of r. For this reason there is a tendency to designate the condiI I till' condenser is imaged in the plane of the object as the I I III "hod," limiting the name" critical illumination" to imag" hi source itself in the object plane.t 1 11"" I, "'I."ils(,IHift fiir wisscnschaftliche Mikroslcopie," volume 10, pages
,NUl), . .

11 h

11111 III l'f1pi'i,\ do not t~kc kindly t? 'this differentiation, especially as , 1111 rlhuu- .uul IIIg-h-pOWl'" illuminarion, Inasmuch as Kohler made no claim 11111111 \ IIrig-illllll'd t hc method :111d it was in common use long before he 11 ""'" I ,llId PI':I('I irnl npplicntion of it. This is cause of some confusion 11I(111111 u iuu f1'01l1 puhlicntions origin:1ling in England.

"i

34

FUNDAiV1ENTAL PRINCIPLES

OF PHOTOMICROGRAPHY

CRITICAL ILLUMINATION

35

If this is done solely on the basis of having a means of differentiation between the two systems of illumination, it is justifiable, but if there be an implied idea that Kohler illumination is not critical, a false impression results, for the lighting is, or can be, equally" critical " whi~hev~r method is used. The true test of critical illumination is that all illum 1nating rays are uniformly disposed about the optic axis and ~f s~fficient aperture entirely (and just) to fill the back lens of the objective.
L

, ~f-l
~ PplA~:..::========tt ~ Light Disc.

Image of

Le

se

.
B

l o~~.

DO,

w.

9<l_'IX ---'1
~;:;;;;:::::::----~fC,-1--~)

of ;1' diameter d located in the plane L - L. The disc is shown turned iround so that we may conceive its surface aspect; actually, it is flat lilt! lying in a plane at right angles to the eye. The same applies to the lluminated area as well. Le is the lamp condenser, with a focus = f, located its focal distance from the plane of the light source. All rays oin anyone point (as at p) on the ~sc, after passing through the ondenser, emerge parallel. Upon rea hing the substage condenser 'C) they are brought to a focus to form an image of the point ham hich they originated in the focal plane of Le. As already exInincd, the position of is soregulated that its focal plane coincides ith the plane of the object. Every other point on the light source I . - e.g., P, - forms its corresponding part of the image; the latter, iwcver, is inverted. Should Pt in the light source be an opaque spot, 'ol'rcsponding dark area would appear in the image and be observed the field of the microscope. Also, if we reduce the diameter of the hr disc - as, for example, by means of an iris diaphragm - the image III also be reduced. The actual size of the light image in tl~ object n ' can be determined on the following basis:

se

image of light; size of light::

focus of

c
Le
Opaque Disc.

",Image of
Condenser

se : focus

of

Le.

Le

FIG. 18. Comparison

of the two methods of obtaining critical illumination and high magnifications

for medium

It is not our purpose, at this time, to analyze the theoretical aspe.cts of the two systems of illumination in their relation to image formation throuzh interference maxima, but it is important that the fundamental differences between them be understood by the practical photomicrographer, that he may know which to use under varying :Giperating conditions. To this end, let us follow out the course of some of the rays from the light source to the object plane, under each system. In Ficure I SA we have two pairs of limiting rays plotted, und r the setup usually known as critical illumination, anal?gou~ to Figl~r' I6A. The light source we will assume as an evenly illuminated dISC

s the focus of a substage condenser of N.A. I -40 is extremely r, the diameter of the illuminated area is small. This means that nrire field of low-power objectives will not be covered unless ns are available to increase the size of the illuminating source. irh this setup, if a small black disc were placed in front of conr Le, as ,at x in Figure ISB, it would have no effect on the image he illuminating disc (except for a slight reduction in the total . lint of light), for the rest of the condenser area would still function (11'111 the image. nsidcr now the second case, illustrated in Figure I The only . in the arrangement is that the light condenser, Le, has been I a little farther from the light source than its principal focus (t) 11its distal focus, no longer at infinity, lies in the plane of the subdiaphragm, sd. The conjugate foci are now fe and fe', respecI . The substage diaphragm is located near the back focal plane I ..Ihstagc condc.nscr. Rays from a point in the light source passhl'Ough condenser Le, after forming animage of the point in the IIUI'CC, ill the plane sd, cross and continue on through condenser 111 '1' rit r 11 'arly parallel, to form disc of light in the plane of the '. Thus, even If the light source were not a disc of appreciable

Se.

FU'DAMENTAL

PRINCIPLES

OF PHOTOMICROGRAPHY

CRITICAL ILLUMINATION

37

diameter, but a theoretical point, a relatively large disc of illumination would be projected on the plane of the object. ., As the diameter of the light source is increased, each pomt ori 1tS surface projects its disc of illumin~t~on .on the. objec.t plane, all.superimposed on each other. Irregulannes m the l.nt~ns.lty of the light at different points, or- completely opaque areas within It, would make no difference on the uniformity of the illuminated area. On the other hand, should. an opaque disc be placed in front o.f condenser LC, as at x in Figure 18D, it would show as a black area m the field of the microscope, thus proving that the illuminated disc !orf!led in th~ pla11:e of the object is an image of the condenser, LC, which ISevenly 111um1nated by the light source, whatever the nature of the latter ~ay be. The diameter of the illuminated area is proportionate to the diameter of condenser LC approximately as the focus of the substage condenser is to the lengthof the conju~ate fO~l:s f(*', Thu~ we can reduce. the diameter of the field by placing an ins diaphragm m front of the 11ght condenser, or we can increase it, within limits, by the use of a largerdiameter light condenser. . . In either of these systems, as the focal length of th~ sub~tag~ condenser is a factor in determining the size of the area of illumination on the object plane, it is evident that th~ use of a sub~tage condenser of longer focal length will also serve to increase th~ dl~m~ter o~ the field illuminated, although at the expense of a reduction m 1tSultimate numerical aperture. . ..' If the photomicrographer can master the practical application of these two systems so as to be able to employ either .at will, he need not worry over the fine points of the controversy \~h1ch has be.en 'wage? over the relative merits of each, from a theoretical standpoint, ThIS has to do with the question of the value ?f coheren~ lig~t in the formation of interference maxima, upon which resolution IS postulated to depend.t .
" The absolute relationship is true only under the theoretical condition where the plane of the substage diaphragm lies in the exact rear principal focus of .the co~de?ser, so that rays from the latter emerg.e parallel. ~ecause of mechanical design limitations, this is usually not the case, the diaphragm b.emg located farther from the condenser. Should the distance be equal to 2f, the equatlon becomes: diameter of illuminated area 2f of substage condenser diameter of light condenser = conjugate focus fe' of Le This is more nearly the average condition, but it does !,ot affect ~he gencral rule n.sto the control of the field aperturc by means of the diaphragm 111front of the light condenser. '1' It is axiomatic that only .ohcrcnt light cun produce uucrfcrcn 'C phcnumcnn,

practically all authoritative works where these two methods of Illumination are discussed, the latter, usually referred to as the Kohler method, is described as distinguished by " imaging the light source in Ihe plane of the back lens of the objective," instead of in the plane of he object, as in so-called " critical illumination'!' This method of I iltifying it, while technically correct, is ~ot very satisfactory from a rncrical standpoint, whereas it is easily comprehended by the phrase il11aging the light condenser in the plane of the object." , either of the systems of lighting described is satisfactory for use ilh low magnifications" where it is necessary to cover a large field. 0111 the practical photomicrographer's standpoint, Kohler's greatest
I

In

Substage Condense,' \

--_c__
Light Condenser
Apertu1"e

Aux. Cond.

I~

Plane of "Object

~*-~_

Diaphragm,

Substage Diaphragm

I J

FIG. I9.

Kohler critical illumination

for low magnifications

ribution to the science of critical illumination is his method of ring a large field uniformly, thus meeting a need not present in nrly days when microscopes were used almost solely for visual
uses.

---I

I . setup is as shown in Figure

19. For best performance all the are different from those in use in the medium- and high-power

It t'h<1ngc of phase. relationships. The theory prorounded by Abbe, that resoI " ponds upon and is proportional to the number 0 interference maxima formed I lIlgular aperture of the cone of light picked up by the objective, has been enI hv advocates of critical lighting where the light source is imaged in the plane I "hil'ct. On the other hand, it is objected that coherent light does not result ~11I method, that only the im~ing of the light condenser in the plane of the 1111 provide coherent light. 1hus the latter method should give better resoluhilt this cannot be demonstrated. The subject is highly speculative. For inIt ('1111 be argued that one must specify what particular rays from the light ItIIl~t b - .ohcrcnt, in order to form the interference maxima in the image. If Itl 0\'('1' i hc entire area is involved, it is correct that coherency can result only It 0 cull id Kohlcr 'illulllination where light originating in one point covers I I Iickl. But it can' be argued. that superimposed upon the coherent light from 11 poil1t is other light, coming from an infinite 'number of other points, each lit \I it h ihl'l f, hut 1101with each other. 1111 th(, ot lu-r hand, it is argued that it is only the rays falling on an individual 11till' ohit'ct thlll nlllst h' cnhcrcnt , such <1 ondition can be obtained only by lit li!lht \II\II'l'(' itself Oil the "bill: of i he object. Thus while the light falling

./

FU

DA:.1VIETAL PRINCIPLES

OF PHOTOMICROGRAPHY CRITICAL ILLUMINATION

arranzernent. Condenser Le can be the same but is preferably of shorter focus. In actual practice thi~ is often accomplished by the use of an auxiliary positive lens placed lI1 fro~t .of .the regular l~mp Condenser. Condenser Le is focussed upon an iris diaphragm whlc.h serv~s as an aperture stop. From here, though the rays cou~d c?n~mue directly to the substage condenser in actual practlce I~ IS found advantageous to insert an auxiliary condenser (aux. cond.) In~he system to parallel the rays entering the substage condenser. This latter is a low-power single-lens (often called a spectacle~lens) condenser, located directly beneath the stage. Its focallengtl~ ISsuch that when in this position, the rays from it come. to a focu~ m the plane ~f t~e diaphragm of the low-power photomicrographic lens used WIth It., Whenever the latter is changed to one of another power, a corresponding change is made in the subst~ge condenser; i.e., a battery of ~ve objectives requires a corresponding set of ~ve condensers.. . With this system large areas, up to conslderab~y over an. mch m diameter, can be evenly illuminated. In effect, thI,S.m.ethod IS analogous to that employed i~1the stereo~ticon, .wh~re It IS necessary to illuminate an area approximately four m~hes ~n d~amete.r. . When the three variant methods of illumination WIth transl:nI~te.d light are compared 'with each other, certain similarities. a.nd dISSImIlarities are evident. Let us analyze these, from the standpoint of operation. First, the only differences in th~ setup bet~Teen the two systen:s f~r high-power work lie in the location of the light c?I~denser, ~1:Ich IS nearer the light source in one case tha.n the o~her, and m the posltlon of the field diaphragm, which must be dlrectlx m front of th~ light source in one case and directly in front of the lIght condense~ m the other. The apparatus is identical in either case. The substage diaphragm controls the aperture in both cases.

39

se,

ill th~ second place, a point that is often overlooked is that critical htillg, within the original understanding of the term (when no lamp nndcnser was employed), can be secured 'with the lamp condenser I 'utcd anywhere betnoeen the two limiting conditions. In other ords, these two methods of illumination merge imperceptibly into I -h other, wit.hom any positive l~ne of demarcation, It is impossible , dercc~ any difference I? .resolutIOn, or m the character of the image, 1/ allY intermediare posmon of the lamp condenser. III the technical aspect of the-two systems, there is the difference in I position where the image of the light source is located; in one it is unrcd in the back conjugate focus of the objective; in the other it is 11' thc. b~ck lens of.the objective. F~'O~la practical. tandpoint, howr~this IS a sm~1l dIfference; for while m the latter case the image of light source ISsharply delineated as one looks down the tube with 'yepiece removed, in the other case it is also seen, but is not in sharp

us.

'hen we come to compare low-power Kohler illuminatlon with other two, so far as operation characteristics are concerned, we I that radical differences exist. Chief of these is the change in the I .rions of the diaphragms. The substage diaphragm does not. conI the aperture of the system, but has become a field diaphragm. If OIW add~tional diaphragm is em1?loyed. \ wh~ch is all that is actually ssary), It must be moved.from ItS posinon lI1 the front focal plane h ' lamp condenser (or between that point and the front surface of 'olldenser) to the front conjugate focus for the low-power sys-, . It then -ceases to be the field-limiting diaphragm, becoming inthe aperture diaphragm. The condenser itself must either be id to one of shorter focal length, or moved farther from the

h .n again, the position of the image of the light source does not
I ,'idc
I',

on each point in the object wo~ld be cO~lerent with it~elf, it would not be coherent with respect to any other point m the object. , In view of the fact that photomicrographically both methods appear to YI~I,d e uivalent results it seems possible that both premIses are wrong; that the true answ t I l,q can hI' les somew Ile re.' else . It is the author's personal belief that the . whole.. problem _. " I d d the Abbe theory of resolution retained on the baSISthat It IS not ncccssru ~ ~~ ~~ns~2er the primary source of illumination at all, so far as coherency IS concern 'd, The rays which form interference. maxima and which must ah~'ays be c~herel~t/~" h eeding from the object Itself, which may be consider cd as mcrcl y cxci tI o~~ ~:~~rnal source, This view need not conflict in any wny WIth the fact t!l'lt ,1111' e[citing rays must have an aperture sLlffi~ient to fill the back lens of the objccriv. , There is a very simple explanation for hIS.

with eit.her .of t~e ot.her systems, for it is located in the optical of the objective, i.e., 111 the plane of the diaphragm, when the r IS present.

is thus seen that we have three separate systems, commonly red to by only two designations, "critical illumination" and hlcr illumination." All three are actually" critical" so far as perIIIIll't' IS concerned, and therefore the nomenclature is misleading. It 'rurorc, t h ' method of low-power illumination is decidedly to be III 'd :l~ ()rigiJ~al ,.virh Ktihlcr in its application to microscopy, us hIS COil! rihur ion to rhc other method sometimes ascribed to

40

FUNDAMENTAL

PRINCIPLES

OF PHOTOMICROGRAPHY

him was merely a scientific analysis of its operation, as distinguished from the accepted form of critical lighting. In view of all these factors, it appears logical to limit the designation" Kohler illumination" to the low-power method, describing the others under the older nomenclature, " critical illumination," with the addition of the phrase " imaging the light source," or " imaging the light condenser," as the case may be. Discussion-of the relative advantages of each of these systems from a working standpoint is reserved for Chapter 4. _ It yet remains, on the basis of what has been said for the various methods of illumination, to sum up the illumination requirements essential to the best performance of a photomicrographic outfit. These are: . . ( I) The illuminating rays should be symmetrically disposed about the optic axis of the microscope, and should be capable of entirely filling the area of the back lens of the objective yet not be in excess of this. (2) The diameter of the illuminated area should beample.tocover the entire field of view to be photographed and means should be available to circumscribe it as near as possible to this area. (3) The, entire area illuminated should possess uniform intensity throughout. When these conditions are met, the light is critical, whatever method is employed to achieve the results. ~ To these conditions might be added a fourth, one which is largely of convenience from a practical standpoint. This is, the light should be ample to enable careful focussing to be done on the ground glass, under any conditions of operation, and yet should not be excessive. This is the service condition which determines, more than any other, which system of illumination should be employed. It will be discussed at length in Chapter 4. With a thorough understanding of these basicprinciples of photomicrography, one is qualified to begin practical work with transmitted light. Such modifications of these principles as occur with other types of photomicrography, such as work with incident light, dark field, etc., preferably can be considered in connection with each specific line of work.
0 0

./

Chapter

MODERN

PHOTOMICROGRAPHIC

EQUIPMENT

lis

xnnplete photomicrographic outfit is comprised of three separate the microscope, the camera, and the illumination equipment. pt in a few special instances, each unit is designed so as to be corn- .' I . in itself. The primary reason for this is to obviate need for a .rcnt microscope, when one is already available. The wide div.erin types of illumination at our disposal an~ the corr,espondmg .in personal needs or preferences are responsible for the frequent nrion of the camera and illumination units. onuncrcial outfits are procurable to meet any need as to simplicity, rifcamera, and monetary outlay. They range from small attach.nmcras, supported directly on the microscop~ tube, sans a~l minaring device, through large and comple~e .equlpments .to uni11designs in which the microscope is a specialized model, integral I nnd not usable apart from the rest of the apparatu,s. . I .nsc is not the only item to consider in the selection of eqmp- , i n jther is the size of picture which can be taken; ?oth advantages Iisadvantages may be found ionevery commercial model, large mall, simple and complex. It therefore behooves. t~e ~ould-be omicrographer to become acquainted with the .rm11ta~1.onsa~1d 1 d advantages of each general design. If one 1Sfa~mhar with I own special problems, an intelligent choice of eqmpment can he made. 01 the purpose of disc:lssing the var~ol~scommerical designs av~ilV' can group them into several distinct classes, as follows: '111 id I nrra .hcd cameras. , iimplv vertical cameras. iruplc horizontal-vertical outfits.
0

.!lI'J.{l rcscn rch models, , If contained universal models. 'P' -iul purpose models,

MODERN

PHOTOMICROGRAPHIC

EQUIPME

GROUP 1J

S fALL ATTACHED CAMERAS

43

Group

I -

Small Attached

Cameras

In Group I there are only two commercial models widely used in this country. Both are of foreign manufacture (American firms do not appear to be interested in this line of equipment). The first of these consists of an adaptation of the Leica camera to the microscope through the use of intermediate fittings, supplied by the manufacturer of the Leica (E. Leitz, Inc.). .

FIG. 20.

Lcitz Micro-Ibso Attachment

The Leica, pioneer in the miniature camera field, is of the roll-film type, employing standard 35 mm. motion-picture film; the size of the picture is equal to two frames, i.e., 24 X 36 mm. Pictures taken with the Leica, whether ordinary snapshots or photomicrographs, are neee~sar~ly quite small, and the common practice is to enlarge them for vIewmg. Three different attachment devices are available for the proper association of the Leica with the microscope. The most complete is the Micro-Ibso Attachment illustrated in Figure 20. It is an elaborat (and rather expensive) outfit, including an eyepiece, beam-splitting prism with side telescope for visual examination and focussing, and :1 Compur shutter. Above the shutter is the connection funnel for at tachment of the camera. The lens of the latter must be removed ill order to assemble the Micro-Ibso Attachment to the camera. When mounted on a microscope, ready for use, the outfit appears as in Figul'(' 21. This is the ideal attachment for use when living organisms arc 1 () be photographed, as with it they can be observed right up to the illstant of snapping the picture.

, .F?r ordinary types of photomicrographic work, Leitz supplies the Iiding Focussm~ Attachment shown,in Figure 22. This is a simpler leVIce, butrequires a separate mountmg base and arm such as that of one o~ .the Leica enlargi.hg outfits .. Focussing is accomplished on a f()CUSSl~lg screen located m the plane of the film; the sliding device enblcs either the screen or camera to be moved into position over the microscope. With this outfit, exposure can be made by ~eans of the mmera's focal plane shutter, because the mmera is rigidly supported independently of I, he microscope. This is not the case when he Micro-Ibso Attachment is used, hence the I rovision of the Cornpur shutter in the latter. The third 'alternative mounting attachucnt is a simple Adapter Ring, for connecIOn of the camera directly to the microscope ibc. In this arrangement, the lens of the mera is allowed to remain and focussing is . pmplished by visual means in the microope.and the Leica lens is then set for infin to approximate the visual condition. he only other manufacturer of mioscopical equipment supplying an outfit pted for use with a standard miniature mcra is Zeiss. Their attachment, for use lth their Contax camera, does not differ rnaially from the Leica design when the icro-Ibso Attachment is employed. It is , F' FIG, 21. Micro-Ibso At)\\,11 m 19ure 23.' , , . . . tachment on Microscope 11crographs as taken on motlOn-plcture 11 film by either of these cameras are shown, at full size, inFigure 24. hese small outfits have several distinctive advantages, but also cern disadvantages and limitations. A knowledge of these is desirable the part of a prospective user. Among the good features are: .. (r) Economy of operation. The cost per exposure, when motionturc film is used, is extremely low, ( ) The space required for the entire equipment: is small. It can rorcd almost anywhere when not.in use, and quickly set up whenr wanrcd. Any place where a microscope can be used for visual poses is satisfactory for photomicrographic work as well. (~) The rrcarcst value of the; miniature camera to the science of

r
44
MODER
T

PHOTOMICROGRAPHIC

EQUIPMENT

GROUP

J -

SMALL

ATTACHED

CAIVkERAS

45

FIG. 22.

Leitz Sliding Focussing

Attachment

pl~otomicrog:raphy lies in its application to the photographing of living mlc7"0-orgamsms. The only alternative is the use of motion-pictur eqUIpment, which necessitates an elaborate and expensive setup and is not nearly so flexible in other ways. Motion pictures, even when taken on 35 mm. film, provide a picture limit cl to one frame si? , as compared to the two-frame pictures taken with a minicum. 1 hell they can be used as motion pi rur S onl: in lnr re projection 111:1('hi, 'S,

h .rcas nearly everyone employs 16 mm. film for .noncommercial Il"poses today - a size. entirely inadequate for general photomicroiphic work (4) The roll-film camera is both convenient and rapid when a large i 's of phorornicrographs in sequence, or under identical conditions, , 'quired. . ( ,) If one continually uses a miniature camera for ordinary work, )roficiency' in manipulative technique can be carried over to in!Ill ' photomicrographic . 11"1, when the same 1\1 .ra is employed for rh purposes. (r.) Although the iniI cost of the camera rh its attachment is h. as compared with " simple photomicrohie outfits, if purdsolely for photornirnphic work embracpccial problems, the m can also be used irdinary candid camrode This possibly open up an entirely hobby to its owner. 7) For those wishing I i photomicrographs olor, the miniature 1 m offers by far ~\Osr inexpensive ap1 'h. Kodachrome is rivcly cheap in rnopicture roll film, as . ipurcd with profes1I cut film in the: I" sizes. The great inI mnnifcsrcd in ret cars in .olor' phophy has extended
I ,

hot'oJllil:ro ~raphy as

flu:. l.!'

Zciss Contnx ..Miflcx

MODERN

PHOTOMICROGRAPHIC

EQUIPMENT

GROUP

I -'- SMALL ATTACHED CAMERAS

47

well, making this one of the most important fields for the miniature camera in combination with the microscope. The enormous enlargement to which Kodachrome can be subjected in projection, and the development of special projectors for miniature lantern slides have been largely responsible, and have helped to establish the miniature camera firmly in the photomicrographic field. Contra these advantages, the following considerations should be borne in mind in deciding what type of equipment may be best suited to one's individual problems. ( I) Because of the small size of negative produced, an enlarger is required for the making of the final prints. (If the minicam is already owned and in use, presumably the enlarger is also.) Should it be necessary to purchase the camera especially for photomicrographic work, the cost of the enlarger should be included in the total outlay required. .. (2) Considering the limitations to which this type of equipment is subject, it represents an expensive form of photomicrographic apparatus, if purchased for this purpose alone. The same amount of money, judiciously spent, will provide a far more flexible outfit. ( 3) It is not practical to take, develop, and study single pictures, as can be done when plates or cut films are used. It is possible, should occasion demand, to do this, by wasting some film and going to the bother FIG. 24. Photomicrograph of reloading the unused portion. negative made on motion (4) Various exposures on a single roll canpicture film, by miniature not receive individual treatment, as to type d ti cameras . o f developer (so f t or contrasty), an time of development. It is this possibility of individual treatment of each subject which puts the finishing touches to ideal photomicrographs. (5) The type of emulsion and film characteristics cannot be changed from one picture to the next. All pictures on a roll of film must conform to the limitations of the particular film employed, r 'gardless of whether or not it is the best for the purpose. (6) A small picture size necessitates rapid exposures, thus narrow ing the latitude within which one must work. Fast exposures are ideal under ordinary photographic conditions but in photomicrography the variables - as to nature of sub] er, nature of lighting, amount of

magnification, aperture of system, filter factors, plate or film characcristics, etc. ~ are such that short exposures should be avoided. (7) There is an extreme lack of flexibility in the amount of magnification obtainable, This limitation is present in all photomicrographic .nmeras which have fixed projection distances, regardless of the size of picture taken. It can be compensated for, to some degree,by varia-

FIG.. 25.

Micro Camera Attachment

(Leitz)

ill the amount of enlargement used for the final print, but this r 'suit in excessive enlargement under some conditions. To bring nrivc made on motion-picture film up to a five-inch circle rea five times enlargement. With a given combination of ob, and eyepiece available, should an object be just beyond the size h cun he included in the field of view with a fixed projection dis,Ihe only recourse is to change to a lower magnification that will he entire object to be shown. Yet this combination may be rluu Ihe illlage of the object will only about half fill the negative. III briu r il ro a full five-inch size will require, not a five times en111'111, hUI nearer I '11. rt h same time the objective giving the I illla r' will probahly possess a correspondingly lower numer-

1\'1(lIJER

PIIOTOMICROGRAPHIC

EQUIPMENT

GROUP 2 -

SIMPLE

VERTICAL CAMERAS

49

ical ;.1pcrturc not capable of full resolutior: when its eyepi.ece image ~s further cnlargcd ten times. Where a vanable extension IS present, It . often suffices to reduce slightly the bellows length until the desired ob-; t> . ject is included. For instance, if one were us10g an extens~on glv10g a magnification of 50X, a reduction to 45X or even to 48x might accomplish the desired result; this would be achieved with t.he larger-apertured lens, yielding its correspondingly greater resolution, (8) Except for the purposes of photographing fast-moving, living objects where an intense illumination results in their quick death, and the making of miniature Kodachrome micrographs, the use of roll-film miniature cameras should always be considered as strictly an amateur device. Such cameras cannot take the place of apparatus designed strictly for photomicrographic purposes, for serious research work. (9) From the psychological point of view, there is one final objection to the use of roll-film miniature cameras in photomicrographic work. This lies in the very cheapness of an exposure and the ease with which exposures can be made. The result is that instead of trying to make each XpOsure as nearly perfect as it can be, one is tempted to take a dozen shots of each subject under 'slightly varying conditions, in the hope that one, at least, out of the lot, "ought to be good." This attitude is not conducive to the development of qualified photomicrographers. It is strongly recommended to all using minicams for this class of work that each picture be taken on FIG. 26. Zeiss Miflex Camera the basis that it 'must be good, just as though each shot cost" a dollar p~r " instea? <?fone cent. In addition to the adapters for accommodating the mmiature ca.meras, both Leitz and Zeiss supply simple fixed-f?cus ~ameras wl:lch mount on the same attachments. These are available m several sIzes and use plates or cut. film in place of the. 35.mn~. roll film. ~hc J ci~:t, model is shown in FIgure 25 and the ZCISS111 Figure 26. ZCISS substi

tutes in this model a small ground-glass screen upon which the image can be.focussed and observed, in place of the viewing telescope. These outfits are considerably more flexible for general photomicrographic work than the miniature cameras, because-of the larger pictures which can be taken and the use of individual plates or films to meet each exposure condition. It is regrettable that American manufacturers do not provide an equivalent of these si~ple attachments to meet the particular needs of those who do not reqUlre a separately supported camera. At the same time, it must be admitted that in every instance where a separate camera can be used, it obviates the obj~cionable feature of the microscope's being used to support the entire npparatus, and the cost of the additional support is not appreciably

more.
iroup
2 -

Simple Vertical Cameras

11 manufacturers of microscopical equipment supply out~ts falling irhin this group. Undoubtedly these are the most popular m the enrr range, from the combined standpoints of economy, size of pieire, and simplicity of design. here is not much difference in design between the models put out the various companies. Most of them have more than one style ( m which to make a selection. All the models include the camera, mnted on a vertical rod, which in turn is rigidly secured to a base It. The microscope, in its vertical position, mounts under the camusually located by adjustable pins or straps, on the base. hrce models are supplied by the Spencer Lens Co. Two of these illustrated in Figures 27 and 28. The former (Model #'630) ludcs a bellows-type camera but the base is a simple iron , hence there is no positive association of the microscope and iera. odcl #'645', shown in Figure 2'8, is more elaborate; although the r()scope is mounted on the base plate in a si~ilar manner. This I,) is equipped with a side telescope, but, as. IS nec~ssa,~y when a t .lcscope is employed, the camera has a definite proJectlOn length. I 01' these models accommodate up to 4" x 5". plates or filf!1s. I' nccr's third model, #634, has the base of .#645 and the. adJ.ustable III s of #630, with no side telescope. It IS also larger m SIze, acuuoduring up to 5" x 7" plates. . h ., three models constitute the entire Spencer line of photomiI

50

MODERN

PHOTOMICROGRAPHIC

EQUIPME

GROUP 2 ~ SIMPLE

VERTICAL CAMERAS

SI

crographic cameras. No special illumination system is associated with these cameras, so that the user is free to employ any setup he may desire. The Bausch & Lomb Optical Co. have a much more extensive line of photomicrographic equipment. Their two models which come within this group and correspond to the Spencer line are the Type K

rnmera shown in Figure 29 and the Type J shown in Figure 30. The former takes plates up to. ~i" X 4i"; the latter is much larger, providmg for 5" x 7" plates, when required. The J model is supplied on a

)0'\(1.

28. 'Spencer

Photomicrographic

Camera No. 645 for 4" x 5" Negatives

I plcn

ental baseboard, :on which the illuminating system is also mnrcd, forming an integral part of this equipment. I Ill' Z .iss and Lcitz models falling in this classification are illustrated !,i 'IIr'S 3 I and p. Both of these include the illumination system as inre rrul parr; both ar also available in two sizes, for 9 x 12 cm.

[i'IG.

27.

Spencer PholOllIicrographi("

C:tIlIl'1':!

(I.

(qu

"

.1") and ," x 7" plur

S.

MODER

PHOTOMICROGRAPHIC

EQUIPMENT

GROUP

4-.-

LARGE RESEARCH MODELS

53

Group 4 - Large Resear~~ Models For the research worker who desires the utmost in larger-sized flexible equipment there are two outstanding models. These are the large horizontal-vertical outfits made by Bausch & Lomb and Zeiss. Before the development of ' the Panphor Universal outfit, the Leitz Company also put out a large research model, but they have withdrawn it and now offer only the Panphor. The Bausch & Lomb quipmenr (known as I BVP) is shown in its horizontal position in Figure 34. he large 8" x r a" camera ith its long bellows (40nch extension) and the exme rigidity of the entire FIG, 30, Bausch & Lomb Type J Photornicroiaratus are self-evident. hi E ' . . grap IC qUlpment hen It ISnecessary to emloy the microscope in its vertical position, the camera can be elevated c()rrespond, as illustrated in Figure 35. The Zeiss r 8 x 24 cm. horimal-vertical outfit is similar in general design. It is illustrated in its o positions in Figures 36 and 37. Of interest in this design is the long tical bench (one meter), which provides great flexibility in opera11 and permits the use of other specialized equipment. * ' iorne years ago, the Zeiss Company prided themselves on their large rizontaloutfit, This was built in two parts: the camera, on its indiIu:~ stand, was s~parate from the microscope and optical bench, hich were carried on a second stand. In this way the camera could used either in a straight line with the optical bench, for transmitted hr, or at right angles to it, for metallurgical purposes. This' outfit is I muted in Figure 38. It was finally discontinued because of two seliS drawbacks. One was the possibility of trouble due to vibration
l'hc present war has made it almost impossible to secure German apparatus of 'SPl! .inlly such hi~hly specialized outfits such as this type of camera, which lH'I"l'1' stocked in quantity, but it is shown here, together ~ith othe~ apparatus of 111I111 ruunufucturc, ill order thnr the state of the photomicrographic art may be ,!loSl'I'illl'd, I'l'f{lIrdll'ss of Il'"lpOl':ll'y conditions.
111'1,

FIG. 29.

Bausch & Lomb Type K Photomicrographic

Camera

Group 3 - Simple H orizontal- Vertical Cameras The only firm supplying a model coming u~1der this classi~cation, at the present time, is Bausch & Lomb. Their Type R eqUlpmen~" shown in Figure 33, is similar to the Type J except that t1:e fixed ver~~cal rod in the latter is replaced by one th~t can be swung 111tO the horizontal position, wh.e~ desired. T~1e rmcroscope may then ?e ~scd in its horizontal posltlon, made possible by the presence of t1:e 111ch.na tion joint. The microscope mirror is removed and the entire Optl.C~l~, axis from the licht source to the center of the ground-glass Iocussing '0 . screen, is on a straight, horizontal line. .' For those who desire a less expensive model capab.le of being u.sec111l the horizontal position, Bausch & Lomb supply this c~mera WIthout the supplemental baseboard and optical bench. It IS then called TypeH.

54

MODERl

PHOTOMICROGRAPHIC

EQ-UIPMENT

GROUP

5-

SELF-CONTAINED

"NIVERSAL OUTFITS

55

occurring between the microscope and photographic plate, as they were free to move independently; the other was the ease with which the two parts could get out of alignment, when employed in a straight line. It was also both costly and cumbersome. It now serves as an example of an undesirable design, or " how not to do it." This must not, however, be understood as implying that these outfits are unsatisfactory in operation, for there are still many of them in use in some of our large research laboratories, where they give complete satisfaction. The great flexibility of the research models of the horizontal-vertical type allows of their use with lowpower lenses of all sizes, as well as allowing the opposite extreme, of high magnification. Changes in the type of illumination, accurate alignment of all optical equipment, the use of spe-' cial equipment, and even the employment of the camera for copying, en larging, reducing, and lan tern-slide making, are :111 possible with these out FIG. 31. Zeiss Vertical Standard Photomicrofits. graphic Camera For one who under star:ds the fundamental. opti~s o~ photomicrography and is capable of settmg up any type of illumination system desired, these outfits, COlll plctcly equipped with accessory apparatus, offer the utmost for any and every class of worl , .

Grou~ 5 - Self-Contained
'/

Universal Outfits

Recer:-t years l~a:vewitnessed a r~dical departure in the design of photomicrographic a.pparatus. Unit .outfits are rapidly superseding many of the older, more conventional cameras. The Leitz Company vere the pioneers in this field when they brought out their Panphor' niversal Microscope. They were quickly followed by Zeiss, with their Ultraphot, and in England by the Vick.rs Projection Microscope. So far American firms have not followed this trend toyard a compact universal outfit. . The original purpose in deigning this equipment ap f cars to have been to provide microscope and photomirographic equipment, selfontained and permanently Iigned, which could be used ir every sort of work by .hrricians not possessing a sic knowledge of all the inciples involved in the varFLG. p. Leitz Vertical Photomicrographic us types of work they .~amera, with Microscope and Lamp, in Posiight be called upon to per- tlon
f111.

I'he microscope can be used visually for transparent work with nsmitted light; as a polarizing microscope; with vertical top illumirion, for metals; with oblique top. illumination; as a fluorescence icroscope, for dark field, etc.; and drawing, projection, or photogphy can be performed under each condition. The change-overs the lighting systems required for these various conditions of op,eraon arc: 11provided for in the design of the apparatus. They can be ndc more or less automatically, 'without the possibility of rnisalignI nt or improper setup. . Space requirements for these universal models are small, a 2' x 3'

,\lOllI< 1(,

1'1I0'l'O,\IICROGRAPI-IIC

EQUIPMENT

GROUP

5-

SELF-CONTAINED

UNIVERSAL

OUTFITS

57

table area being ample for any of them. * It must not be assumed, however, that size reduction has brought about a corresponding lowering of the cost, for these universal outfits are anything but inexpensive, especially if purchased with all the attachments necessary for every type of work. The basic model of theLeitz Panphot for general work is illustrated' in Figure 39. It would not be feasible to attempt to show the appear-

FIG. 33.

Bausch & Lornb Horizontal-Vertical

Camera (Type R) in Vertical Position

..-.--~~----

-, I ' of this, or other makes of universal outfits, in all the various set'} of which they are capable. For some work the changes are minor, It for others, one would hardly recognize the combinations as having relation to each other. For work with transmitted light, the arn cmcnt must bc such that the illumination is from below the stage. 11' lOp illumination, mctallography, etc., light enters the various lhuuin.u ors [rom the rear, above the stage. The standard plate size
(1)(:.

FIG. 35.

Bausch & Lomb GBVP'Camera

in Vertical Positi~n .

1+

Hnusch

&

l.omh

PIHI\()lllit"l"ogl'nphil' Outfit,

(;IlVP,

l lo rizuutul )J(1~itioll

IllIpll'\\'d

\\'hl'1I d,is is l'111I1 rust 'd with rhc author's large horizontal-vertical outfit with 0111'1I11'll'roplical benches, requiring :1 floor ~pace about 5' x 9', the 111111' i \1'1r cv idem.

MODERN

PHOTOMICROGRAPHIC

EQUIPMENT

GROUP

.s: SPECIALIZED

PHOTOMICROGRAPHic

APPARATUS'

59

of the Panphot is 3t" X 4t" but Leitz will supply on special order a camera to take 5" x 7" plates. The Zeiss Ultraphot is shown in Figure 40. The most radical difference ~etween this outfit ~nd the Pa~1phot lies in the employment, in the ZeISSmodel, of the main supportmg frame as the lamp housing for the low-voltage lamp, which in the Leitz Panphot is mounted externally, on the right-hand side. Both these outfits can be equipped, when desited, with a supplemental arc lamp, which connects into the regular light train, in place of the low-voltage lamp.

itself weighs considerably more than two hundred pounds. Special cquipments for all typ~s ,of work, including polarized light, etc., are available. Whether future trends in photomicrographic equipment will continue along these lines, to the final elimination of the older research models, is still an open question. For those starting with a universal outfit, it may seem difficult to graduate to the older optical-bench apparatus, but, on the other hand, one who has grown up with the latter'

FIG. 36.

Zeiss Horizontal-Vertical

Camera in Horizontal Position

.Undoubt~dly th.e most radical departure in microscopic and photomIcrog~aph~c eqUIpmen~ is ~he Vickers Projection Microscope * shown m FIgure 41. It IS an inverted form of microscope, with the stage on top and the objectives pointing upward, as in the Le Chatelier metallurgical microscope. The reason for this appears to be two-: fold - fi~st, it is primarily i~1tended for a metallographic outfit, and second, It employs a reflecting system to extend the projection distance and enabl: one to vie~ the imag~ at a comfor~able ar:g1e, iooking downward. LIke the Leitz and Zeiss models, It reqUIres marked changes in the setup for accomplishing various results. Alteration of the projection distance is effected by varying the position of the reflecting mirror, each inch of movement being equal to two inches in the projection distance. An ingenious but complicated arrangement is provided for automatically changing the mirror angle and maintain ing the plate normal to the projection axis as the projection distance is varied. This is shown diagrammatically in Figure 42. The me chanical construction is very rugged; it is claimed that objects up t () fifty pounds in weight can b supported by the stage. The ourlit
Manufactured
by Cooke,
Ti'Clllf(hllll\

FIG.

37. Zeiss Horizontal- Vertical Camera in Vertical Position


.'

ould probably never be content WIth even the n~ost thoroughly [nipped universal microscope. It is, ill the last analysis, largely a matr (If personal inc;lination. '
run] (/ -

SjJeciaiizedPhoto711icfegraphic

Apparatus

&

SillllllS,

Yorl , I'I\I-\'I:II\(I.

Certain types of photomicrographic work are so highly specialized to I' '~Iuir' radically different setups, as contrasted WIth th~ more I ntionnl lines of work. In some cases these setups may involve ) rra cquipm mr and sli rhr modifications of standard outfits to

60

MODERN

PHOTOMICROGRAPHIC

EQUIPMENT

EQ IPMENT

I-OR

LOW-POWER

PHOTOMACROGRAPHY

6r

adapt the ~atter to the class of service required. In others, a complete outfit, designed specifically for one line of work, is necessary. . The recent trend on the part of manufacturers, especially those puttmg out the universal outfits, is to provide the necessary attachments and ~ccessory ~ppar~tus ar~anged to mount on, or at least utilize, some portIOn of their baSIC eqUIpment. For some classes of work this is quite satisfactory, but in others it is obviously only a makeshift.

its OWN. merits. No hard and fast rule can be made to cover every condition. In gen~ral, however, it can be stated that where only one particular line of work is contemplated, or where the extra expense ?f duplicate equipment, or sp~ce lin:itations are not factors, more sa~Isfaction will result from havmg unit apparatus for each highly specialized line of work

"

FIG. 38. Zeiss Two Unit Outfit.

An older model, now discontinued

Among the types of work which can be considered as fallinz within this general classification ~an be mentioned: metallurgical p110tomicr.ography:; photog~aphy m the ultra-violet region; colloidal work WIth the sh~ ultr~-mlcroscope; spectrographic and fluorescence micros- ' coPy; motIOn-pIcture photomicrography; and stereoscopic photomicrography. . Special appara~us, as well as complete specialized equipments, to handle all these kinds of photographic work are made by various manufact~rers. As each type of work will be discussed later, with the techniques ad~ptable to each, it will save duplication to cover both the apparatus Itself and the problems arisinz in its use at the same tim . Figures and descriptions illustrative of this specialized equipment will accordingly be found in Chapter 5. I~ procuri~g out~ts for specific purposes, where there exists a choi . , of either ~ unit eqUIpment, designed to do a ingle type of work in rh . best possible manner, or adaptations of universal outfits through rh . employment of accessory eqUlpment, ea h case must be consi lcrcd Oil

FIG. 39,

Leitz Panphot

1l1i/1l11ent for Low-Power


Ill'

Photomacrography

I"hotomicrography enters 'a region o~ magnification not l~su~lly re-d, or employed, for visual wor~{ wlt.h the compound ~lcroscop~. his includes the range from full-SIze pIctures, up to. possibly 20 di1)1 t rs, where the average lowest-power combination of objective I 'yepiece usually starts. '. . .. 'hus a s rics of photomicrograpluc lenses, designed to, work withI ". epic .cs, must be ava,ilable. Th~se function as ordinary photoIphic lenses, the only (11fT erence being that they are computed so \I t hci r ,longer 'on jugare focus is at the ba~k of the lens, the shorter t hcin r ill front. The nvcra re lens of this type, regardless of focal

62

MODERN

PHOTOMICROGRAPHIC

EQUIPMENT

EQUIPMENT

FOR LOW-POWER ,I

PHOTOMACROGRAPHY

length, usually works at a full aperture of f+5, or approximately a numerical aperture of .10. They are then provided with iris diaphragms, for reducing the aperture and increasing the depth of focus. The field of such lenses is quite flat over the entire working area, in this respect differing materially from the curved field so characteristic of compound microscopes when ordinary eyepieces are employed. The entire range of magnification involved cannot be covered by any single lens; a battery of several is necessary, although .with a complete series and a long bellows it is possible to extend the magnification well beyond that provided by a lowpower objective in combination with a low-power eyepiece. Each manufacturer designates his lens series by a special trade name, and the specific focal lengths of the lenses comprising the series vary somewhat with different manufacturers, although not to any material, extent. The Bausch & Lomb series is designated by the name Micro- T essar. The set includes four lenses, 72 mm. (2t"), 48 mm. '(2"),32 mm. (d"), and 16 mm. (t") focus, respectively. Figure 43 shows the complete set, together with the corresponding condensers required for transmittedlight work, and the barrel mount' FIG, 40. Zeiss Ultraphot employed when a microscope is nor used. l- or still lower magnification than that provided by the 72 111111. Micro-Tcssar, the series can be supplemented by the regular TI B Tcs sa rs, working at f': 6.3. These are made in various focal lengths extending down to 4~".

FIG, 41.

Vickcr's

Projection

Microscope

101>1';1{, PIIOTOJ\IICROGRAPHIC

EQUIPMENT

EQUIP

![ENT FOR LOW-POWER

PHOTOiVIACROGRAPHY

65

S _-F=:!,!~-L-F=~

SS

The Spencer Lens Co, low-power photomicrographic re~ses are known as Micro- T eleplars. The four lenses in the set are similar in equivalent focus to the Bausch & Lomb set except that the shortest focus lens is 24 mm. (I") instead of I 6 mm. (t"). The Spencer set is" shown in Figure 44. Leitz lenses of this type are called Summars and Micro Summars.

FIG. 43. Bausch & Lomb Micro-Tessars with Camera Lens Board Mount and Special Low Power Condensers '

The set includes five tenses, 100 mm., 80 mm., 65 mm., 42 mm., and mm. focus. In addition to these highly corrected lenses, Leitz also puts out a 'series not so well, corrected, at a lower price. These ~re designated Milars. In, equivalent focus they correspond to the Sum-, mars except in the case of the two shorter-feeds lenses, which are 40 mill. and 30 mm. in the Milar series, instead of 42 mm. and 35 mm., as in the Micro Sumrnars. ' For many years Planars were standard with the Zeiss Company. he were made in r oornm. (4"),75 mm. (3"),50 mm. (2"),35 111111. (f!"),and 2OlTIm. 0") sizesall working at f.a.c. Recently this series of five lenses has been replaced by a new series, known as lVlikrotars. 1 h 're are eight lenses in this series; instead of all working at a uniform aperture, the aperture is progr ssivcly increased in the shorter-

~5

MODERN PHOTOMICROGRAPHIC f()ClIS

EQUIPMENT

EQUIPME

T-FOR

LOW-POWER PATA

PHOTOMACROGRAPHY

lenses and reduced in the longest-focus lens. Increase in the f rat ill as the focal length decreases is consistent with standard practice on all other microscope objectives and enables much higher magnifi-, cut ions to be employed without introducing empty magnification. '1 hl' optical data on the 1VIikrotars, as given by the Zeiss Co., is on pa ,\' 67. Six of the lenses in the Mikrotar series are illustrated in Fig1II'l' -I- ' With all the manufacturers, it is standard practice to equip such

OPTICAL

OF THE MIKROTARS

Focal length cm.

Aperture, - ratio

Numerical aperture

Theoretical lower limit of useful magnification 4

Scale of Approx. reproduction' size of field for I meter disof view ranee of image from principal mm. point 5 6

f: 1.6
I

I5 2

f: 2'3

0'3 I 021 015


O I

.I'55X I09X

99: I 66: I 49:


I

3'5 5

"

f:32

78X
55X

7
IS 20 30 60 . 80

:'5

3)'

" . f:4'5

( 32: I 2I : I

l 16: I
0.08 40X { 10: I 7=1

-+}

f: 6'3

FIG.

44.

Spencer Micro-Teleplats

Il'll . " where the size permits, with the Royal Society screw thread so I hili I h 'V Ilia), be mounted on the microscope just as though they were IIhl '!iVl'S. This is feasible up to about 60 mm. (f: 4.5) lenses. Beyond 1111 I Will Ihe diameter of the lenses increases rapidly. Unless the miI Ill' is ()f the large-tube type and provided with means for carry11 I h ' 11I1Ig'focus lenses, the latter cannot be used on the microscope I Ill, hili I1ll1St he mounted directly to the front board of the photo11\ III l'lpllic (,:II11Cr<1. The old Zciss method of mounting the larg , II 011 Ihl' hig 11Ih' microscope is vcry practical, because it proI 0 JlIlI '11 addit iona] space between the srnge and the lenses, It I I 111'11 [uunc] (Fi rurc 4t'l) scr 'wing into t he !"Of> of the till} ,:tl1d I1 III d III till' hOllOlll I() uccouuuodnrc the I:trgcsr-di~lI11et 'I' I ns. As I 11111 ,I i materially sllml er t hall till' t uhc itself, t he lens 'S ar t lIus

carried ~vithin the tube, entirely out of sight, providing arradditional space of a couple of inches betw.een the lens and stage, .for ?-ny given position of the tube. The nosepIece and bottom .tube r111S'111to which it mounts must, of course, be removed when this setup IS employed. To do this quickly, a large tube sliding chang.er is avai.lable. 'In the later models of stands, where 'the enure tube IS made interchangeable for monocular and binocular purposes, the funnel js available as a funnel tube accessory. In all cases where low-power photomicrographic lenses are' employed in transmitted-light work, on transparent objects, it.isnecessary to use a substage condenser suited to the particular.lens in question, in order to provide critical lighting of the full field being covered. Ea.ch manufacturer accordingly provides such condensers as standard eqUIpment. Those of Bausch & Lornb are included in Figut'e 43: These condensers are not required for incident illumination of opaque objects. '.' For many purposes, especially when the longer-focus . lenses are used, a microscope is not necessary; it may even be. a handIcap. The omission. of the microscope means that the photomicrographic lens~s must be mounted directly on the lens board of the camera. To t~1IS end, most manufacturers provide some type of fixture, preferably WIth focussing means incorporated in it, for .carrying t1:-e lens_~s OI~the lens' hoard. HallS .h & Lomh's device for this purpose 1S seen 111 FIgure 43.

Ml

MOlDlmN PIIOTOMICROGRAPHIC

EQUIP ME

ILLUMINATION

EQUIPMENT

":or vcrt~cal can~e~as whe~e the lens is within easy reach of the hand, while ~H1C IS cxammmg ~he Image on the ground glass, a simple screw Iocussing arrangement IS ample. When, however, a long, horizontal c.uueru IS employed, some extension-rod device for distant control hCCOlllCS nec~ssary. Zeiss' lens-board focussing mount for use with Iht, largc horizontal outfits is shown in Figure 47. Rotation of the

FIG. 45. Zeiss Series of Microtars,

in partial section

side spindle, by means of the distant control rod, moves the lens I h I'OU rh the operation of a spiral thread on the lens mount.

llltnnination Equipment
Illumination problems involved in photomicrography are quite dii .. Icrcnr fr0111those occurring with visual work alone. The principles of correct illumination are not at issue; they are the same in both. The issue is sol~ly th.e in.tensity of the light source required. For photography thc illumination must be ample to enable accurate focussing to ~)c.done, even when color filters of very Iow transmission character isncs arc employed, and also to keep cxposure times within reasonable lilllit.s, A ligh.t that is i~\cal in intensity for visual work will he found 1"0 ~H,,'ll~ pra~'t1C.::1IIy no IIllagc Oil th ' ground glass, 'V '11 ar " t .n inch PI'Olt'CtlO1l dismncc, when :1 grecn screen with n filler factor around III

is intr.oduced i? the light ~ain. On the other hand, when a light source s~lfficI~nt to gl~e a good Image on the ground glass is being used, the visual Image WIll appear of dazzling intensity and the eye should not be placed at the .,-epoint, as injury to the eye migH! result. This is why special sources of illumination are supplied for photographic pur~ poses. In the early days of photornicrography, three brilliant sources of light were considered suitable: the oxy-hydrogen limelight for those without ..electricity, and the arc lamp and the Nernst lamp for those who were fortunate enough to have electric current. The limelight is now a relic of the past; so also, to a great extent, is the ernst lamp. It 'is rather surprising that some enterprising manufacturer has not developed a ihotornicrographic lamp upon the crnst principle, .for it is a beautiful light source, comparable to any of the special high-intensity lamps on the market. Its only drawback is the preliminary heating required for the glowers, but this could be FIG. 46. Zeiss Funnel for Microtars, nsily overcome by proper design. shown on Microscope , The arc lamp, though alwaysac# xpted as a suitable light' source, has had its objectionable features, not he/least of which is the inherent tendency of the arc to wander, shiftin!:! its position out of the optic axis. Proper design has overcome this objection, so that today the arc lamp is thought of first when maximum intensity of illumination must be provided. . The simplest form of the arc lamp is the hand-feed type, usually of about five amperes current' capacity when used on direct current and t '11 ampcres on alternating current. If the lamps are properly designed tl~cy 'an be made to work equally well on either d.c. or a.c. but the hi rhcr operating amperage is n ccssary with the. latter, as the effective

\ 10111,11" I'1I0TO,\ IICROGRAPHIC

EQUIPMENT ILLUMINATION EQUIPMENT

71
.

intensity is ('OITt'spolldingly lower, because the voltage passes through ZCI:O twice in each cycle. As hand-feed lamps are not very satisfactory for photomicrographic work, all manufacturers supply, at a somewhat higher cost, mechanical-feed lamps. The lamps are mounted in suitable housings, properly ventilated, and so designed that extraneous light in the room is kept to a minimum. .(This, of course, is equally true of all types of lamps F,G. 47 Zeiss Focussing designed for microscopic work.) Bausch Mount on Lens Board. A L b' . 1 " & om s autornanc arc amp IS Illustrated long focussing rod is at. F' cached to the spindle shown m 19ure 48. The condenser is supplied at the right hand side. as an integral part of this lamp. It can be furnished with or without a cooling c.dl, and arranged to mount on a separate portable base or on standard riders to fit the Bausch & Lomb optical benches. Th~ Zei~s mechanical-feed arc lamp is shown in Figure 49. This lamp IS deSIgned to mount only on the Zeiss triangular optical bench, and as all Zeiss condensers, water-cooling cells, etc., mount on the bench on separare riders, the lamp is not equipped with any of these accessories. The Zeiss lamp is unique in one respect - the horizontal carbon is of very 1110111 diameter (5 mm.) and I orcd. This construction 'oruplctcly eliminates wand 'ring of the arc. The small diameter is compensated for hy a more rapid movement of the horizontal carbon and a 8 I1 greater car b on 1 IIIUC engt 1 1, FIG. 4 . Bausch & Lornb Automatic Arc Lamp as compared to the larger-diameter vertical carbon. The arc lamps of other manufacturers follow the general design of onc or the other of these two illustrated. Because arc lamps may have to be used on either direct or alr :l'IulIill " current, standard pracncc is to supply suitable rhcosrnts for use with t hcm. "I his is the only method of op 'rating arc lal11psOil direct cur

nr, with alternating

current, however, a step-d.ow~ trans~ormer can

h . employed to advantage. As these are not ord~nanly available from hc microscope manufacturers, they are often d1fficu.lt t~ secur~, but
I'C

desirable .where the heat from the rheostat - which IS considera-

hi . - is best avoided.
Ithough the use of arc lamps is essential for some classes of service, or the greater part of ordinary photomicrographic work the light is

FIG. 49. Zeiss Automatic Arc Lamp

ccssarily intense and frequently very objectionable. The develof other sources of concentrated high-intensity illumination in ,':nt years has provided the microscopist with a wide range .of lamps, . Olll which he may choose according to the type of work bemg d.or:e. I,hc lamps available can ~e ~rouped into four gen~ral ~ypes: Minia11 tungsten arc lamps within vacuum bulbs, which includes Sl1Ch 11 known forms as the Point-e-lite, the Bausch & Lomb Tungsten , crc., low-voltage; high-alTIp~rage, ribbon-filament lamps; co?cen- . td Iilamcnr projection lamps; and photoflood lamps. SUItable 11 ings:lr nee 'ssa,.)' with all of these; but where the type of base h whit" lamps .irc cquipp .d is th same, onc type of lamp can usu11

III 'm

MODERN

PHOTOMICROGRAPHlC

EQUIPMENT

ILLUMINATIO

EQUIPMENT

73

ally be substituted for another, when desired. It should be pointed out, however, that lamps in the first two groups require external additional equipment in the form of starting resistances, controls, transformers, etc., to render them operative, so that mere exchange of one type of lamp for another will usually not suffice. Lamps of the vacuum arc type are relatively high priced, especially when the accessory equipment is included. This is also 'true, to a lesser extent, of low-voltage ribbon-filament lamps. The Bausch & Lomb Tungsten arc lamp is shown in Figure 50. Concentrated-filament projection bulbs are usually designed to operate directly on I re-volt circuits, so that no extra transformer or rheostat is required.' These come, however, in three different basings - the large Mogul base, the standard Edison base, and the bayonet-lock base. The latter is standard for small motion-picture projectors, in various FIG, 50, Bausch inrensities up to 750 watts; these constitute an ideal & Lomb Tungsten light source, although they have not yet been so Arc Lamp widely adopted for photomicrographic work as they deserve to be. The same is true of photoflood bulbs. These will undoubtedly soon be put to use in photomicrography, as they constitute one of the least expensive sources of intense illumination,

lump.

ork can be done. ' This also saves current and extends the life of the For this reason projection and photoflo~d lamps are inclu~ed I available modern eqUlpment because, assummg the lamp housing umished by the manufacturer to be adequate, the particular light

FIG.

51.

Zeiss Electric

Sodium Lamp

1 uomicrographer, 1 tipped' with the

FIG, 51,

Bausch & Lornb Qunrrz Mcrcury-vnpor

AI"

Lamp wirl:

Itrn-,"iolel

Sl'lIlJ1

tree installed within it is readily subject to change, at the will of the or lamp housings alone may be purchased and preferred type of lamp. , .' . )11 addition. to these light sources for ordinary photomicrographic I oscs, there are others available for specific purposes. Two espeI v, nrcof importance - thequartz mercury-v~por arc.an~ the elecodium lamp. Both of these are rather expenSive, but ~ndlspensable .rt nin types of work, Mercury-vapor lamps.are available fo: opnun on either direct or alternating current. For photomicrophie purposcs thcy arc housed in a mal:ner similar to ar.c lamps, 'and be equipped with portable bases or riders f.or the optical benches,

OJl'

gr ar advantage both projection bulbs nnd photoflood 1:1111]1.' possess over other typtS is the possibility of switching in :1 r(.'sist :1I1('l'to Jowcr till' illlt'lIsity to :\ poillt where till' prcliminnrv visual

I sired,
\\IS

-h & 1,()lllh's Illl'l'cllry-vapor lamp is illustrated in Figure 5 I. '111')'V:lpOl'1:lIlI]>S find their -hicf use in ultra-viol'!' and fluorcs-

74

MODERN PHOTOMICROGRAPHIC

EQUIPMENT

MI9CELLANEOUS

ACCESSORY EQUIPME

75

cence ph?tog.raph~, but are also of value wh.en a sharp band in the green ~eglOn.ls desired, ~s the 546 mIAspe~tr~lline is so extremely intense, It provides a beautiful monochromatic lizht source in this reo-ion The sodium lamp, the Zeiss model of which is illustrated in Ffgur~ 52, serves the same purpose as the mercury-vapor lamp, in providing a monochromatic light of great intensity, but in the yellow, through the double D line of the solar spectrum at 589 mu. No filter is needed with the sodium lamp, but one must always be used with the mercuryvapor lamp, for passing either the 365 mu or 546 mIAline, but not both at the same time as would happen if no filter were employed. All of these light sources find their primary application in the photography . of ~ransl?are.nt ~bjects, by transmitted Illumm~on, lIght or dark field. Only the most intense of them are suitable for vertical illumination - metall~graphy, etc. The same is true of low-power incident illumination with oblique light. For this purpose an ideal arrangement is an arc lamp with focussing condenser, mounted on a portable flood stand and adjustable as t9 height, say from 3 to 5 feet. . Such equipment does not seem to be standard with the microscope manufacturers, but the floor stands are easily procurable and it is a sirn pIe matter to mount the arc or oth ir high~intensity lamp, complete in its h?usll1g, ~n the adjustable support. FIgure 53 Illustrates the general idea. A very practical illuminating de vice for low-I)()wer photomacro r [<[G. 53. re Lamp 011 Floor Stand raphy with .ir le!" transmitted or ill

I nr light, which can also be used for copying and reducing.as t~e , TO Stage of Zeiss, shown in Figure 54. For opaque work the ll1?Iluul top lamps are used, as desired, the object being suppor~ed, ~Ith uiruble background, on the flat glass stage. When the object IS to photographed by transmitted light, il1umir:ati~n is effected by lus located under the glass stage, each of which IS separately con- . III.d. . )1 her forms of illuminating devices for low-power :"orkare .avail11 from the various manufacturers. The Macro-Ring Illuminator I I,ight Box of Leitz are shown in Figure 55

{lsccllaneous

Accessory

i':q7tip'l11ent

In addition to the cam optical benches, and

I t sources, which con~(- the essentials of a Itlllicrographic outfit, t of the larger manuIll' .rs supply numerous surics, some of which ilmost as important for iin classes of work as more basic elements. f of these is the cool"H, an absolute essen\ h '11 arc lamps are em-. U. Some other lamps in heat rays are also to cause damage to 01' objects unless a . lin r eel] is inserted in

li rhl

'ia 11 rru - when the ,II,d Kiihl 'I' illuminai -mploycd, for hi rh nifll':tl ions,

rrain..

This

is

coolin ,. cdl
lit III

I :tll'S
'Oil

of

:1

rlass

MODERN PHO'fOMICROGRAPHIC

EQUIPME

MISC~LLANEOUS

ACCESSORY

EQUIP

1ENT

77

tainer with parallel sides of plane surface glass, capable of holding water or an aqueous solution of salt, alum, or other chemical having a strong absorption for infra-red rays. The Zeiss form, used either singly or in pairs, as required, is shown on its individual dual rider, in Figure 56. This is a very practical design. The body of the container is made of heavy porcelain, the sides of which are ground flat. I~lain glass. disl~s are held in place on each side by means of clamping rIngs; leaking IS prevented by the use of rubber packing rincs. The cells can be easily taken apart for cleaning or to replace a b~oken or scratched glass disk. For ultra-violet work the glass disks can be. replaced by a pair made from fused clear quartz. These latter, however, are quite expensive. In addition to their function for cooling purposes, cooling cells can also be ultilized for holding aqueous calor filter solutions, if desired. (Information about such solutions is given in Chapter +) For most purposes, however, commercial filters are prefer-' able. For this reason, manufacturers provide some means, which' can be inserted in the light train, for holding such filters. The filters are available in"two forms, the 'IV ratten dyed gelatin films mounted between glass, as supplied by the Eastman Kodak Co., and solid colored glass filters, manufactured by Schott & Gen, J ena. The two-inch-square size is ample for almost all photomicrographic work, although larger sizes are available at a higher price. eutral tint and heatabsorbing glass filters are supplied by Bausch & Lamb. These are available in different densities. ~n special equipment for use in the light tram on the large outfits, the Zeiss Co. leads all others, on account of the e111~ ployrnent of the one-meter optical bench 1'1(1. H. L itz Macro Ring Iland the unit design of each piece of appahuniuat or and Lighr Box rams. It must be noted, however, that th a Ivent of the universal lrruphot has 1ll01'(.' or less placed such apparatlls Oil the " special" list, to rcrhcr wit h all forllls (!f rh .ir l:1rger research phororuicro rraphic mod .ls. This

special equipment includes special condensing lenses from 7" in diarpiter down, of various focal lengths; centering condensers; condens.rs with associated iris diaphragms; large and small iris diaphragms; ohject supporting tables; shutters; filter holders; cooling cells of various types; polarizing prism, etc. Each of these is provided with its own individual 'rider; also, separate riders are provided on which the photomicrographer may mount, any special equipment of his own. To myone working with one of the large outfits, this represents flexibility in the extreme, but, at the same time, it assumes a theoretical knowledge, -npable of setting up and using the various pieces of apparatus, which I often not in evidence. ' nother piece of auxiliary apparatus supplied by Zeiss, and which hould -be made available by every manufacturer of photomicro~'aphic equipment, is the M~:tltiplicator Back, shown in Figure 57. his back fits on the camera m place of the regular plateholder, and h latter then mounts on the Multiplicator. It ...-""",,--.' --"""'1 nnhlcs the plateholder to be shifted, throughout I 'ngth, across the centerline of the camera. h in, by means of metal masks, mounted in the uucra in front of the Multiplicator, having nings of predetermined widths, a 'whole series 'posures of an identical field can be taken at ious exposure times, or with various filters in tight train, for determining ideal exposure I dirions. This is a valuable adjunct for elimi111guesswork and making direct comparisons. IS impractical to attempt to cover all the IOUSpieces of equipment available. Espe11 is It impossible to do justice to all the aux'quipment designed for use with universal trs such as the Panphot and Ultraphot. FIG. 56. Zeiss CoolI 11the underlying principles ofeach type of ing Cells on Optical Bench Rider J toniicrographic work are, once compreI d, the information in the manufacturer's 10 ucs is usually ample to guide one as to' whether a particular I Ill' Iliece of apparatus is essential for his peculiar needs. With i 1':11 cnowlcdgc of these needs before purchase. of an outfit; the 1IIIIItiol1given should suffice to enable a proper selection of equipI t III he made. pt for the universal ourfirs, where the microscope is an integral he apparatus, all other photomicrographic cameras assume

MODERN PHOTOMICROGRAPHIC EQ IPME TT

MISCELLAl

EOUS

ACCESSORY

EQUIPME

TT

79

ownership of a separate microscope which is to be adapted to the outfit. In the majority of cases, the microscope will be used for visual purposes as well. When the microscope is already on hand and a new one is not contemplated, it must be used, in spite of any inherent limitations it may

..

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o

0..

'" b'n '" .~ E 8


o

.
.

FIG. 57. Zeiss Multiplicator

Back

possess. When the microscope is to be purchased with the rest of rh ' photomicrographic equipment, there are some points relative to the instrument that must be considered. I f it is to be used exclusively for photography, the special photo Illicrographic microscope of Bausch & Lomb (Figure 58) will he found to III .cr very need. But for combined visual and photograph] ' work or h .r mod 'Is arc mor suitable. Spt't'i:t1 art .nrion should b given to the following .onditions: (I) l l iirh quality work at hi 'h rnn mifiC:1rions rcquir 'S a h ':1V , ri rid t.uul, \\'ith t hc hest (im' motion Iocussin r mechanism possible. !'11ll'

80

MODERN: PHOTOMI<;,ROGRAPHIC EQUIPMENT

motions on low-priced stands are often quite coarse for critical work. (2) Low-power work with large camera-type lenses of greater focal length than 60 mm. requires a large tube stand, and ample distance between lens and stage. For this purpose those stands in which the stage is adjustable by rack and pinion are preferable. (3) For high-power work, a slow-motion mechanical stage is essential, as otherwise a minute object cannot be centered accurately. At 2000 diameters, a utouement of the object 1/1 oootb of an inch displaces it two inches on the ground glass! (4) Proper angular orientation of the object with respect to the edges of the picture requires a rotating stage, especially' in high-power work. (5) The question of the optics of the microscope should be settled on the basis of the degree of perfection desired in the photomicrograph. Although for visual work achromatic obje :ti ves will usually prove adequate, and for ordinary routine photo- ' micrography will also serve, provided they are not forced in their performance, they are far outclassed for photography by the apochromats. These ~atter shoul~ always be used when the highest possible results arc desired. For Visual work, compensating eyepieces are required when apochrornats are used, but these are not ideal for photomicrographic work because of the extreme curvature of the field attendant upon their use. To produce a flat field with apoclfromats, the Zciss Co. developed the series of Homal eyepieces, which give beautiful results. These are quite expensive, require a complete set of sevcrul to cover the entire low-, medium-, and high-power fields, ana cannot be used for visual purposes. evertheless, their usefulness will flllly repay all they cost. The Bausch & Lomb Co. put out a similar . " which they call" Amphiplans." The substage condenser should also he of a higher order than the ordinary Abbe two- or three-lens 'ypcs. For most purposes, an aplanatic 1-4 N.A. condenser suffices, hut some critical work calls for one that is chromatically as well as sphcrically corrected. I- inally, for best results, some means should be provided for individually cenrering each objective with the condenser. It makes no di IT crcnce, however, whether this be accomplished by cenreriqg the' condenser or the individual objectives. . . Attention to all these minute details in the selection of a rnicroscop , will not be found wasted.

Chapter 3 HOMEMADE EQUIPMENT /

The possibility of employing horr:emade equip~ent for taking photomicrographs probably suggests itself at some tim,e or o.t~er to 'vcry owner of a microscope, or at least to those ~h? m addition to the microscope also possess a camera of some description. aturally the question arises, " If the two could be attached together somehow, -ould not a picture be taken with th~ ~ombin,ati?n?". In many cases only a lack of knowledge as to the optic~l pnncirle~ involved, 07' the l!'Oper way to proceed with the mechanical aSSOCiatiOnof the ml~ro'opc and camera, prevents the putting of the idea into actual ~ract1ce. III theory, at least, every owner of a microscope and cam~ra (regardI ss of.ihe type) does possess the" makings ". of a photor~llcr?~raphic outfit, capable of taking practical pictures, since the avallabllity of a Ii hr source (the third essential) of some sort can be assumed. The rimary requisite is that they be aligned so ~hat the projected im~ge om the microscope can enter the lens openl11g of the caI?era, while, tT,C same time, all other light is prevented from entenng. In the n -rical application of this requirement several minor factors. must taken into consideration if satisfactory results are to be achieved. l1lollg them are the following: . . . ( I) Some means must be provided for retammg the l:1lcroscope an.d mcra in proper relationship. This necessitates some simple mechani[mounting for them. . " ( ) The microscope and camera may be mounted either vertically / horizontally, with equally satisfactory results. The type ofcam~ra I l rclarive ease of mounting them together are usually the determinfactors, unless the nature of the objects to be phot?gr~ph~d nec,est 'S a horizontal stage (e.g., anything mounted In liquid which (Hllll not remain in position if the slidewere tu~ned on edge). . ( ) '1 he preferred arrangement calls for the removal of the camera t whenever it is feasible to do so, and the use of a ground glass for Issing the image on the plane of the film or plate. Where this canbe done b)Z means of the camera itself, as in a roll-film camera

81

HOMEMADE
to

EQUIPMENT

HOMEMADE

EQUIPMENT

(with plate and film-pack cameras it is possible), a focussing tube with a ground glass at one end may be employed. The tube, when mounted over the microscope, should measure the same distance from the eye lens of the eyepiece to the ground glass as that from the eye lens and film, when the camera is in position. Focussing is then accomplished I> closing the shutter of the camera, removing the latter, substituting for it the focussing tube (which need not be larger than, an inch in dimuercr), and focussing the image sharply on the ground glass. Then, wi: hout disturbing the microscope, the tube is removed, the camera 111>1'1 il urcd and the exposure made. (4) Where the camera lens cannot be removed, it is satisfactory to IIluw it to remain; with cameras which can be equipped with a ground lass in the focal plane, the procedure is not different from that where Ih ' lens is removed. But for cameras where the focussing cannot. be dune in this manner, the focussing must be done by visual observation in the microscope with the camera removed, after which the camera, set at infinity focus, is placed in position and the exposure made. Assuming a normal eye, which in the position of relaxation normally [ocusscs at infinity (thus being, in effect, a camera focussed at infinity when looking through the microscope), it is apparent that when the visual focus is correct, the substitution of any other .amera, also focusscd for infinity, will not change the condition of focus, and the image should be sharp on the film. nfortunately, this method does not always provide ideal results in every case, for two reasons. In the first place, many eyes are not normal, so that when the image is visually sharp it is not actually in focus for infinity. That this is true can be demonstrated by someone with a normal eye observing the image as focussed by one with an abnormal eye, when it will be found that some alteration must be made in the adjustment to produce a sharp image for the normal eye. A second complication arises in the case of fixed-focus cameras, which are not s ,t at the exact focus for infinity, but at a compromise focal position known as the hypcrfocal distance, so as to get near-by objects in focus also. The r suiting image will not be ideal with such cameras, al.; though it may SlI ffice for many purposes."
To provide n personal touch illustrative of this method of taking phoromicru !(l"IIphs, the author can relate his induction into the field of photomicrograph)" when 11 11lnd jllst Oil!, of. high school he bou~hl hi,S first microscop , before the 1lI1"1lof I Ill" ('1'111 Ill}. I Ih" I 11 Ill' Ill' "'as also talong plcllln.:s with a fixed [ocus \\" " J\" 1)(1\ l'llIlIl'l"Il; it \\,11, 1101 SlII'prisil1g', therefore. lh:1I lhl' id ':1 of ("olll\)il1illg IIH' 1l1il"l"ONI'0IH' IIlHll'III11l'1"II 10 11111'pil'III1'I'S N(HIIl slIggl'stl'd iiself'. 111his srunll h0l111' 10WI1 ill WI'~ll'1'l1

(5) 'Some practical'means,.such as that,described on page. 82, must he provided to assure a light-tight connection between the Il11CrOSCope nnd camera. If the adjacent surfaces of each are flat and parallel, even n thick felt washer may suffice. . Although the mere juxtaposition of microscope aI:d camera 10 pI'oper relation. will su~ce to take plc~ures, onc~ the thrill of,producIllg even a mediocre micrograph this means has been e~penenc~d, .a lcsire for a more elaborate setup ISalmost sure to result, since the 11mInrions of so simple a combination will soon become apparent. One viII want to go further, with ,more elabo:ate apparatus of s?me sort .: The extensive line of eqmpment available for photomIcrographIc I urposes,as turned out by various manufacturers, and the consequent rear range in price, provide an:ple accommodation f~r almost ev.ery pocketbook, :when one does decide tot~ke up rhotomlc,rogra~hf 10 a more pretentlOllS manner. For the ~rdma~y mlcroscoplst there IS the ddcd thrill of possessing and working WIth stan.dardlzed appar~tus. t might seem, therefore, that little need ~hould anse for the la1>onous 'otlstruction of elaborate homemade eqmpment, but such an assumpion is not wholly verified by facts. . . There are two groups of in~ividuals ~ot satisfied WIth such prosaIC 11 ans 'of acquiring a photomIcrographIc outfit. Strangely en~ugh, om the standpoint of financial rating, som~ of these are as w,Ide\y pnrated as the antipodes, ?th~rs are scattered. 111 between. P~actlca~ly 11. hovvever, have one thing 111common: ,th.ey are m~cha11lcally 111lined and derive real pleasure from thebuilding of their own appara-

?y

US.

mong the author's f:'iends are perhaps a dozen who are well luipped with the best microscopes ~~at money: can b~y. Tbey also 1 \I' workshops, with fine .lathes, ~illll~g mach111e~,dnll presses, and I like, where much of their spare time ISspent. It ISno wonder, then,
1111 vlvania, without access to any books on the subject and without another individj() in the town also possessing a microscope,.he was entirely unaware that this had .ever 1\ done before. But to think was to act. The procedure was to take the m~crolip' und camera out of doors whc:re bright sunl,ight was available, ~ocus the mlcr?~Ipl' visunll y, an,d arrange the ,Posltlon of the Illlrro,r so that when It ,was rotated IJ1 pivoted trunnions, direct sunlight would pa~s across the field of view. The box. 11\1"11 was then balanced on the top of the microscope, the shutter opened and the rror rotated I nstchc position of illumination by direct sunlight. The effect was one 11uuntancous exposure (possibly of the order of 1/50t~ of a second). The shutter thvn c1osl'd and the 1)latc developed, By acc,idem, a perfec~y timed, negative tlltl'" I1l1d, ("()nsidl'rill~ rue qunl itv of the cheap objective 'Y,lth which the microscope l'l)lIipPl'd, 11 fairly shnrp pi .turc resulted. Obviously, what one fool has done,

he

'1111do,"

HOMEMADE EQUIPMENT

"

HOMEMADE EQUIPMENT

-that their favorite microscope is not the elaborate product of some w?r1d-renowned manufacturer, but one that is homemade in every detail, the lenses only excepted. And it is but natural that some of these who are also interested in photomicrography have constructed the ncccssary outfit to enable them to take photomicrographs. It does not matter that the ulti.mate C?st has far exceeded, that entailed by therur('ha~c of commercial eqUIpment. Often, after the completion 0 the ~Hltht and the tak~ng of a.few photomicrographs to demonstrate that It actually works, Its practical value to the maker it about nil; it was not huilr primarily for use, but for the joy of making it and possessing it nfrerward, as an example of the maker's mechanical skill. e On th? other hand, there are numerous microscopisrs who have a real longmg to take ~lPphotography, in a serious way, with the microsC()I~ebut are financially unable to purchase even the most inexpensive OUtfit on the market. The fact that there is a limited demand for photomicrographic apparatus of all types, especially as compared to other products more or less in universal use, results in a-relatively high c.ost of manufacture. The co~t of mar~{eting is also increased proportionately. These facts explain why SImple apparatus, which inherently does .no~appea~ to be more than a $25 value, may bear a price tag of five or SIXtl~nes ~11lS amount. ,They also explain why -'nany individuals, who by pmchmg 'and scrapmg might gradually accumulate a sum of $ 2 5 toward the gratifying of an ambition, either lose all interest whcn they discover that it would cost them not $25 but $ I 25, or else conclude to evade the high costs by constructing the apparatus them- ' selves . . 'I :here are,l1Undre~s of ~laborate homema.de photomicrographic out. (~ts III p~'actlcal us~ I? t~11~ country. ~ossIbly there would be many tunes t!lIS.number If In.d~vlduals knew Just how to go about designing and building them. It ISm the hope of providing such information that rh . present chapter is included. Important outcomes ~f popul~rizing hO~1emade equipment arc the dcv~I~)pment of greater mter~s~ ,m photomicrography, the training of :ld.dltlOnal.wor1{~rs, the possibility of publicizing new discoveries ill Illl~~roscoplcal sCI~nces through the use of photomicrographs, and :111 ultimate increase m the demand for commercial outfits: All of rh 'se arc decidedly worth while. At the ,outset certai~ matters should hc definitely settled h [orc nr tl.I:.lIl"gOI1~g ahca~l, With the. construction of any equipment. Till' term cqurpmcnr does not Imply m 'rely a .amcra placed at tilt' c 't'

85

end of a microscope with a light in front of the mirror, such as already explained. It is true that such combination does embody the use of the three essentials of a photomicrographic outfit, and pictures can be secured in this manner. Very little ingenuity is required to assemble such apparatus; the ingenuity will have to be manifested in the taking of pictures worth showing to anyone as examples of one's ability in things microscopical. photomicrographic outfit should be conceived of as a piece of quipment, comprising a camera, a mounting base for a microscope, nnd an illuminating system, all so assembled as to constitute a unit "herein all the parts are definitely related to each other in a fixed manlier, and capable of being moved about without interfering with the tup. '. The construction of a good ahotomicrographic outfit requires coniderable mechanical ingenuity, If one does not possess such ability, it I hotter to enlist the aid of a friend who does, even if he knows nothing If the theory of operation. The theory can or should be, in eve.y case, upplied by.:the microscopist, and he should not undertake any designn or construction work until he understands all that must be accomI lishcd by the apparatus, ' The following are some of the important factors governing the ultinnrc design. arurally, many of these are identical with those which nusr he considered in the selection of the proper equipment when it is I he..purchased. It is to be assumed that anyone planning to construct I mucmade equipment along the lines suggested in this chapter will also udy carefully the commercial models.available, and the particular admrugcs in each design, before undertaking any construction work. What is the maximum size of photomicrograph which might be luil'cd?, ' ( ) \Vhat is the general type of work contemplated - transparent, \l11I " metallurgical, motion picture" or other? ' ( ~) Is one type of work to be done exclusively or is it possible that genII horornicrographic work of all sorts will be required of the apparatus? , '( ) \Vill any work be done in which thematerial to be photographed 11 Ill' :,1 a liq-uid medium> ) Are In .ilitics available for constructing rnanyof the parts of metal, 11111'1 1 he en 1 i r ' nu rf t hc made almost exclusively of wood? fI) Ilo\\' large 'an the complete apparatus be, 'to be used in the space lnhlc 1'01' it?
(I)

HOMEMADE EQUIPMENT

HOMEMADE

EQUIPMENT

(7) Should it be so designed that it can be easily taken apart for storing; ftcr use? . (8) What kind of illumination is best suited to the conditions under which it is to be used? (C) Is it likely to be used with more than one kind of microscope? ( I ( Are the conditions under which it is 'expected to be used unusual' ill :lily respect? _ ( I I) Will the work be mostly with Iow, medium, or high powers? (I ) ls there any likelihood of the optical bench unit and camera being lIsl'd for purposes other than photomicrographic - e.g., copying, enlargill " reducing, lantern-slide making, etc.?
:l

I .ct us analyze the bearing of each of these questions on the final design. ( I) 1 he size of the largest photomicrograph which is to be taken has a direct bearing on the camera required. The size and type of camera, in turn, are tied up with other points for consideration. Atthough there is now a marked tendericy toward the use of small cameras, with a subsequent enlarging of the prints, as in mini cam work, many potential photomicrographers would not have facilities available for enlarging. To have this part of the work done comaiercially, d la corner drugstore, is unthinkable. The use of small films will, therefore, in all probability be limited to those who already possess some make of miniature camera and are content merely to mount it on-the top of a vertical microscope. Even here it is imperative that SOD) means be available for determining when the image is in focus on the film. Cameras of this type which are equipped to take plateholders and arc provided with a ground glass for focussing have a decided advan rage in this respect. , , In general, in homemade equipment, it can be assumed that 'a more )rctentiol1s camera size will be desired. Usually there is a definite re nrionship between film or plate size and the bellows extension avail uhlc. This seriously limits the use of small cameras for photornicro graphic purposes. The best commercial type is unquestionably tlu: double-extension view cameras, for here we obtain the maximum pos sib le bellows length. They are not made in sizes smaller than 4 x inches, but, on the other hand, if serious work is to be undertaken, Ihis is the smallest pl.ate size that should be considered. In su .h a uucrn, ' hy the use of kits, 3i" x 4~" plates or films can be used for rCIlCJ':lI' ";OI'k, .YCI rhc larger size is nvailahlc should it be required.

This same principle applies to the still larger sizes,s" x 7", M',' X 8t", and 8" x 10", and in' addition the increased bellows length associated vith these sizes is decidedly worth while. View cameras of these tizcs, as well as long-bellows cameras of the Poco and Premo styles, are usually available in second hand condition, at very cheap prices. Even ,hen they are not very serviceable for regular photographic work, bemuse of lack of rigidity, they can 'still be .made to serve in a photomicrographic outfit, provided that the .bellows does not leak. ' On the whole, wherever possible, preference should be given to the nmcra with the largest plate size and bellows extension available, for his is the most flexible. It need not be more expensive to operate, beuusc, with the aid of kits, small plates can be used. ( 2) The matter of the particular type of work to be done is closely I .d up with the degree of flexibility required in the apparatus. Should n 'talJography or low-power opaque photography alone be contemlured, the apparatus best suited for each purpose would not only difr from each other but would be entirely different from that required 1'Iy for transparent photomicrography. All three of these could satncrorily employ either the horizontal or vertical type of camera, allough the former is more flexible and also less complicated in the matI' of securing and maintaining axial alignment of light for transparent 1II'1e On the other hand, whereas transparent work requires the op11 bench to be aligned with the optical axis of the microscope, in a rizrmtal outfit, metallographic photomicrography calls for all optical I -h at right angles to the microscope and camera, in order to project hr into the vertical illuminator. * ( J) If only one kind of photomicrographic work is contemplated, I uully pays to design the equipment solely with this end in view. I ,foreknowledge of broader requirements for it will enable modifiIOnsto-be made in the design to adapt it to any needs. For instance, h ample facilities for producing special apparatus, it is possible, as ftl h ' described later, to employ an optical bench planned for t~ansIII work in metallographic work as well. In other words, by \'in~ :.J1l the problems to be met, and all the ways in which the ap\tllS .IS to be used, it is possible to design it so that it will be prac!-Iy

.mivcrsal.
Particles suspended in liquid, or objects which cannot be tur-ned
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I hi

lilllllllll~l'I\phi('

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must hc-und rsrood to apply to the use of ordinary microscopes In the 11 10 cl '1'11rnctnllographic outfits, such as illustrated roo, I he sniuc J>l'in .iplc applies, but the optic axis of the objective Iht\ illuminut ion t rnin. .

11111 IJi,\I>\IW F<)l'11'IF T ,. on their sides or properly supported in SlI .h positions, must h photo graphed with the microscope in a vertical position. Therefore, fill this type of work exclusively, a vertically designcd outfit is the Silllpbl solution. Where such work may be only occasional, there are two :d rcrnative designs. The first is a combination horizontal-vertical ourtir, somewhat like that illustrated in Figure 33. If the outfit is CO I1St 1'\1(':I cd of wood, this is not ideal, although possible. The alternative is ;\ hori zonta] design in which the camera can be elevated, still in a horizontal position, to above the level of the eyepiece, with the microscope in ;J vc..rtical position and so located that illumination can be effected \ it h Ihe mirror, Then a right-angle prism, such as that illustrated in FiguJ'(' ()(i, 'an be mounted over the eyepiece to project the image into t lu: camera. Methods of accomplishing both these ends will be given lat '1', (s) Unquestionably, outfits constructed entirely of meta-l, along rh ' lines of simple commercial models, are superior in strength to, and It'~s cumbersome than, those made of wood. But few of those most likcl. to undertake the production of homemade photomicrographic app:; ratus will have machine-shop facilities available for making metalparrs. The alternative lies in the use of wood throughout, or at least for all but the simplest parts. Suggested designs, covering both metal ;11111 wood constructions, will be offered later. (6) Space considerations mayor may not be a vital ~ctor. When one has available a large room in which the outfit can be set up, it \\ ill u.sllally be found to pay to be generous in every dimension. largl' size. camera, a very long bellows, a sturdy table for support, and a lOll!' opt ical bench between microscope and lamp (one meter is an idl':d It'll rlh) will give greater flexibility than can be secured with a SIlI;lII, ''1I1III1I1t'1lI mounting within a couple of square feet. But often Cllll' 11111 I \\ 1111in n small apartment room which serves a dozen 'other pur 10 I \\ ell. 111 such a case, beggars cannot be choosers; minintnrc ItllI po ihlv (If the vertical type (as requiring less floor space) 'I 1111 I 10 serve, Yet even these can be designed so as to do hi rh I ClI 1 , , . of 111 'corollaries of the small-space outfit is often a n 'cd fOI ""1111,.11111 111 -ntirc outfit after every use, so that 'it may be Sl()l'('d III I h 'I wccn times. When the dining room or kitclu-n 1111',1\1i,l1.a I~all bedr?om, happens to be the on I. P\.I('( , \ 01'1,illS discouraging, to say the least, and Iikcl y I Cld(' 1 of allY but a born optimist to have to sI' .nd a 1:11' ,'r p:111~ I 11 time in setting up and taking down the :Ippal':ll\l~,

HH

110,110 1\111'I C)IJlI'110'I' " fill thought given to rhc design of:1 simple equip~1ent which can IIIlpat'l,cd and assembled completely 111 fivc or ten minutes, all re~dy \I " then easily dismantled and stored away at the end of an evemng tun, will marcrially aid in solving this problem. . H) III this electrical age, the majority of ~vo~k~rs WIll have c~rrent ,I ihlc, so that the problem of light. source. ISlimited to the p,arncular of lamp to be used. But occa~lOns anse where makeshifts must , orrcd to. These may be anyth1l1g from kerosene lamps, gas, batli rhts, to daylight, or even direct sunlight. The apparatus must I I rued accordingly, . ) Occasionally a microscopist may use more ~han one mlcrosC?pe hi work, the second one being a petrographical or metallurgical 1 I. These may not be the same height fr?m the base? when used 111 horiwntal position; the bas~s may: be of dI~erent design; ?r the re.lpositions of the base, optical aXIS,and Imrr~r may be dlfferer~t 111 . -rt ical position. If both are to be us~d m.terc.hang~abl'y 111a 111I1i 'rographic outfit, this must be borne 111 mind m deslgr:,1I1gthe

hI.
'( ) .Somctimes a photomicrographi~ outfit is want.ed for a very

, uul condition, An illustration of this can be found 111 the work of 1 ue \Varren P. Bentley, whose pictures of snow crystals a~e known
'I' t he world. His apparatus was homernade and very SImply ~e1 for this o.t~especific purpose. It was employed at an open W1l1~II\ a small room in which the temperature was kept below J2 F. O\lI'l'C of illumination was daylight. His set~lp .was so arranged \ I'i.l'tllre of a snowflake could be exposed with m a few se.conds I It: f1:1l<c was placed up~~ the slide .. Use of .an outfit 111the Oil an exploring expedition, etc., might be Cited as other ex0

I ,

..

I'

the work contemplated is in t~1elow-, medi~lm-, or I owcr regions, or all three, may have a beanng on tl~e des~gn best for the purpose. Certainly the highe:- t?e I?agmfi~at101:, the ,i rid must be the construction for the elimination of vibration, ) Tl..fucr that a horizontal outfit embraces a camera and optical I ill uli rruucnr suggests the possibility of utilizing it for purposes , ,1',111 ptlO!olllit'l'ographic. If such use is contemplated, the SIze of IIlIt'l':I is imporrnnr. n 8" X 10" camera ca~1be emp~oyed for , '1l11l1'gl'11I 'IllS I)y I(~,iding the plateholder.wlth enlarging paper H 01':1pl.uc .ind providing means on th~ optical bench for mountI '" .uivc, with illumination from behind.

I,) \ Vhcrhcr

HOMEMADE

EQUIPMENT ,.

SUGGESTED BASIC DESIGNS

It is possible that other local conditions or special requirements may be present in addition to these which have been suggested, but enough have been given to illustrate the line of approach to determining the best ultimate design. So much for generalities; the next step is a consideration of specific design and basic requirements. ()IlC of the first requisites for successful photomicrography is freedOI1l from vibration. This is very essential with high magnification, und .orrcspondingly less so as the magnification is reduced. An appreci.u ion of the way in which vibration affects the photographic image aids in the working out of designs tending to minimize it. It is not the presencc of vibration per se, introduced through the floor or walls of the room in which the apparatus is used, that is objectionable. It is . only such vibration as causes a shift in the image on the plate during the time of exposure, that deteriorates tlie image. As a rule, the exp()~ur~s employed in photomic.rogr~l?hy are of appreciable length. \ Vith instantaneous exposures vibration has no effect. A shift of the image on the plate can be produced only by a relative flexing movement between the microscope and the camera. If the support on \~hich th.e .microscope. is mounted also supports the camertand is suffi<:Icntly.r~gld that no vibratory movement can occur which will change ~he posltlon of tl:e p~oto~raphic plate with relation to the image being impressed upon It, vibration of the complete unit as a whole bas 110 I "": T.he other extr~me, of mounting ~he camera as a separate and dis IIllt:t Ul11t from the mIcroscope a~d optical bench, is the worst possible (~'slgn.l~nless they are to be used 111 a room absolutely free from vibra 111l1l. I he m~ralls, whatever type of photomicrographic equipment is planned, vertl~al or horizontal, provide a rigid unit suppon for the ':\11 le1':\:1I1d.mlc~oscope. If a discontinuity must exist anywhere, so fa~' as vibration IS concerned, let it be between the optical bench and 1111(' roscopc. It must not b~ thought that th~re is no need for a fixed relationship between the optical bench and microscope, for this is desirable in order 10 rcra in the optical p~rts i.n perfect alignment and adjusrmcnr wit h en .h ot~ler. SJl~ple VIbration, however, of a magnitude capable of destroying definition when movement is possible between the micro scope and camera, will hav no ffccr on rh illumination of t hc spl'ci 1I.ltll,110matter how 11I~I<:h I1I()Vel11 .nr llIay result between t he illuminn ~1()1l syst cm and rhc III1(TOSCOPl', and conscqucm ly will not :IffecI I Ill' IIl1are.

Suggested

Basic Designs

er

With a view to illustrating what can be accomplished with very little expense and yet provide for high-quality results, we suggest .a few. designs. Let us suppose that a horizontal outfit of generous dlme~slOns is desired, but facilities for working in metal are not available. Wooden construction is the alternative. Our three fundamental pieces ofapparatu~, the m~c~oscope, camera, and source of light being procured, we are m a posltlon to ~eter~l1lne 'ertain essential dimensions - the length of the baseboard, Its WIdth, and the height of the optical center. In all proI:>ability the size of the .amera will be the determining factor in the WIdth of the baseboard. Probably 'about 8 to 10 inches will accommod~te. the largest camera likely to be used. Even with a :1-" x 5" ca~1era It IS preferable to employ-a wide base. If, however, size and weight are factors~ one can.get IIlong with a 6-inch width, especially if.the bellows length ISnot excepionai. The length of the baseboard ISgoverned by the sum o~the exreme bellows length, plus the overall length of the microsc.ope in the horizontal position, plus the dimension allowed for. the optical bench Iorrion, and in addition, if possi~le, an extra 10 to 1.2 l,nches t? allow the limen! to slide back from the mIcroscope for preliminary visual work. he space required for the optical bench is that which will carry the llllll!?, two condensers, filter holder, and a cooling cell, should !his b.e I 'cssary. These can be squeezed into about 18 inches, but twice this Ilire is preferable. he baseboard should be made from straight-grained wood (almost I kind will serve) free from cracks and well seasoned .. Fryed~m ")\11 warping is essential; and a thickness .of a-bout I ~ to It inches aids crially. If thinner wood must be used l~can be reinforced along ~he torn with heavy cleats; On th~ top SIde ~f the baseboard, gu:de ~il s, about ~-inch square, extendmg the entire length, are screwed III g the outsideedges. Looked adrom the end, the completed baser \ appears as in Figure 59 . Str<ps_ . Gwide should be taken that t he ~~ "~ lrh between the guide 'strips is 0 BaseboariL same from end to .end so that ~-'-----. ------T\---l\ 11,) '\ fitted between them will ~ .l+--Reinforcing StripS->-.:. J I I the cntir distance without FIG. 59. End View of Baseboard ling and without apparent . J lay . .'I.'his is all thcr~ is to the bascb~ard, although It should I I' I ' finished and varnish .d. Also, a PICCCof felt cloth can be

HOMEMADE

E~UIPME

TT

SUGGESTED BASIC DESIGNS

93

glued to the bottom if it- is to be used on a table where scratching might result. This design should not be used without support under the entire length, as it can be easily affected by vibration. With this form of baseboard, it is intended that every piece of apparatus used on it be mounted on individual blocks capable of sliding between the guide strips. The height of the mounting blocks for the various apparatus must be such that the optical axis of each will coincide with all the others. All apparatus must also be carefully aligned
/Iii Metal
Plate
I"

Iris ~/.Dia;phralJm ~ (Kohler Illumination)


"

!I

dinary porcelain socket. The socket is mO~.1l1ted on .its w~oden slide block so that the center of the lamp globe IS111 the optical aXIS. Phot~flood lamps are not only in~xpensive bu.t ha~e the added ad~antage of a frosted bulb. A higher-priced alternative, 111 a clear bulb, ISone of the tubular concentrated-filament lamps such as those used with 16 mm. motion-picture projectors. A 500-,watt .lamp is.aJ~ple for a~lwork,ex'ept dark field. If dark-field photomicrographic work IS <;ontemplated, an arc lamp is prefera?~e over all others. It sho~11dbe arranged o mount interchangeably WIth the lamp used for ?rd111~ry.work; arc lamps are not so desirable for general transparent illumination, as the .
Commercial Angles

Fillere (to accO'I1tOdate . Filters)

Optical Axis

o
o o

FIG. 61. Filter' Holder FIG. 60. Simple Mounting for Condensers

from side to side. '\iV orkers with unusual ingenuity can provide means for vertical and horizontal adjustments if they desire. It is preferable that the camera be mounted on an individual sliding block long enough to accommodate the full bellows extension. The foclIssing means of the camera itself should provide for changes in the bellows length. With this arrangement, the entire camera can be p~lshed bac~{ Ol~tof the way so as to make the necessary preliminary isual cxarmnanon and select the proper field to be photographed. After once assembling the camera on its sliding block, it need never he removed. This, however, is not true of the microscope, as it will probably be required much of the time for visual work. For this rea S(!11 the ~11icrosc~pe cannot be screwed to its block, but must be pro vidcd WIth locatmg means so that the foot will always rest in its exact position. A means of clamping it readily in position must also bc pro vided, as a micro cope is not particularly stable in the horizontal posi tion and could be readily dislodged by a jar. As a li rhr source, where electric current is available, probably one of the simplest is an ordinary photoflood lamp mounted upri rht in an or

posure is too short for proper control. W~en more. t~an on~ light mtcc is to be employed, care should be taken.111the on~111aldes~gn of , apparatus that the height of the optical aXISbe sufhciently high to -onunodate the taller lamp. . . means of controlling the intensity of photoflood lamps for VIsual nmination purposes will be described l~ter. _ . '. I etween the lamp mounting and the mIcroscope WIll be mounted the : s~ory apparatus of the optical bench. This may include the lamp ndcnscr, secondary centermg condenser, a filter holder, and a .c~ol'ell. The condensers, in particular, constitute about the most diffiI mounting problem if the constr~cti~r: is to be limi.ted to? wooden i n, largely on account of the desirability of centenng adjustments. the other hand, making the condenser mounts of metal offe~s very le difficulty to theaverage practical individual. A. square pIece ?f 16" sheer metal with a circular hole in the center, slightly smaller 111 111 .rcr than the condensers (which should be at least 3 inches in di1\1'1 '1'), is the support for the latter. Thre~ small cleats will h?ld the I I us 'r in place, while two vertical slots 111 the botton: provide adI. menus for mounting the metal plate to an angle-Iron support,

94

HOMEMADE

EQU1PMENT

SUGGESTED BASIC DESIGNS

9S

T~le angle iron, in turn, mounts on the wooden block support. Details of construction are shown in Figure 60. The same design can be worked out in wood also, but naturally the construction is more awkward. " , The ,usual size of filters employed for photomicrographic purposes IS two inches square. A holder to accommodate these can be easily constructed along the lines suggested in Figure 6 I. As different types of filters are of different thicknesses, and as, also, it frequently happens that more than one filter must be employed to provide a definite color band, places for two or three should be provided. These can then ac-

~----,
I I

I I Optic I

Axis

G'uidc (Strip

Baseboard

Support FIG, 62,

Block

Design of Horizont~l

Outfit

commodate at least two thick filters or one thin and two tl*k, if three slots arc used. If the author's r~lethod ?f illUl:nination, as described on page 192, is employed, no coolmg cell ISrequired for a photoflood or soo-watt conecru rarcd-filament lamp. With an arc lamp, however, one must al\\'.I~S he used. Preferably the cooling cell should be such a one as Ihosl' furnished by the manufacturers of photomicrographic equipIII 'Ill, of which there are several types on the market. In lieu of one of t h 'S " however, it is possible to employ a large, flat-sided bottle, about oil ' inch to two inches thick. Care must be exercised in the selection of 11 'h:l bottle, it is usually possible to find one with clear, flat sides. Distortion of the rays from the lamp condenser should be kept to a miniI III 1111.

It is desirable to provide a metal shield around the lamp to reduce the extl:anco,us light in the room. This may be easily constructed of gal vnnizcd iron. hole, about I { inches in diameter, is cut 'in the.line of the optical axis. In the author's system of illumination, an iris diu phr:lgm, with a corresponding maximum opening, should be mounted Oil the outside of the lamp housing, over the hole. Also, if a clear 1:1I1Ip

bulb is used, a ground or opal glass should be mou~ted over the h?le, on the inside. This latter can be so made as to be easily removable m case illumination with the clear bulb is required in order to shorten an ~xposure. With Kohler illumination, a field diaphragm should be mounted on the lamp condens~r supporting p~a~e, ~s shown, by ?otted lines in Figure 60. The dimension of the lr~s dlaphrag~ ,m this case must be considerably greater than that required when It IS used on rheIamp housing. The completed outfit as described is shown in Fig,ure 62., Where metal construction is possible, in place of wood, th.e pn~ary difference lies in the adaptation of metal parts to the same basic design. The baseboard is replaced by a metal I-beam of the prop~r length and a?out 2t to 3 inches high, depending upon the overall SIze of the eqUlpment. This l-beam must in turn be mounted on a baseboard, but the latter need not be so heavy as in the case'of the all-wood design: The board and I-beam are shown in section in Figure 63. Considerable care must be exercised in the selection of the l-beam. It must be perfectly straight' from end to end. In a.ddition, it should also be c~refully finished by hand filing or otherwise, on the top and top edges, m order that the apparatus riders may slide freely, , ' With ingenuity, the riders may be designed ~o,be ea~ily removable, et .~apable of bein~ clamped s0.as ~o be ve~y ngld, ~lgures 64A and U show simple designs along this line. le IS a good Idea not only t.o mark the positions of each piece of apparat~s on the baseboard or Optl'nl bench (especially if it is necessary to' dismantle the ~pparatus e~ch imc It is used), but also to provide some means of clamping everything olidly to its support as well. In ,t~is way al~ parts of t~e apparatus, , from camera to light source, are rigidly associated at all times. ~robibly a wooden sub-base for the camera, l~~~nted on the :uetal riders, ill be found to provide the greatest ~e:ublhty of operatlon: It may Jso be desirable to employ wooden spacmg blocks for the Imcroscope md lamp mounting, if there is any material difference in the height of heir optical centers. \ Vhcrc a .small amount of money can be spared for a portion of h equipment, a material improvement over either design illustrated vould be secured by the purchase of a standard optical ?~n~h such as hose put out by most-of the manufacturers, That of Zeiss, ~llustrated III Fi rurc 3(), is triangular in shape, and one meter long. With such a

HOMEMADE

EQUIP~ENT

~
PROVISION FOR EXTRA-LONG BELLOWS

97
&c. (Wood Support Blocks fasten to Plate)

Smooth finish to) and edges


1"
11

2"2 or 3 Steel I ; beam Tapped Holes ~--4+-=---=..c::.:;

bench, one can then obtain a sole plate for mounting the microscope, and riders carrying~ all types of accessory apparatus lamp houses, condensers, cooling cells, filter hold=

B
A
1" 4 Carriage

Pillar for Condenser. \

Bolt & Wing Nut

SUpp01t Block Baseboard

ers, etc. These items need not be FIG. 63. Base and Optical Bench of purchased all at once, as rnakeshifrs Metal Construction may be made to suffice until an entire . . outfit can be gradually accumulated. These desIgns 111 wood and metal will be found ideal so far as a horizontal outfi~ is concerned. Mo~eover, they can be readily dismantled for st~rage III a closet when not III use. Their greatest limitation is that the mIcroscope. cannot be used in the vertical position. If both hori~ontal an~ vertical work are contemplated a modification can be made 111 the design, s~mewbat along the lines suggested in Figure 65. Here the camera po~tlOnof the baseboard is separate from that for the microscope and optical bench but is hinged to it so that it can be used either hon~ontally or eleva~ed.to t1:1~ vertical position. Side braces must be provIde? for th.e vertical posinon to provide for rigidity. The microscope ",:,111 req.t1lre a separate. bas~ plate for each position, as in one case the optl~al aXISof .the illumination apparatus must coinci~ with that of the IllIcros~ope 111 the horizontal position, while in the other it must cor,respond wI~h the cen.ter of the microscope mirror. Should ~ strictly v~rtIc~l camera be desired, the design can still be ulon r the Im~s shown ~n FIgure 65, but no provision need be made for :ll:l'~)(III~I()d.atll1~ the mIcroscope and camera in the horizontal position. JllIs will sllnp.hfy th: constr~ction materially, 11 alterna~Ive design, which allows the microscope to be used in 'Ith.er the h~>rJzontal.or v:ertical position, although the camera is always hor~i'..ontal, IS shown 111 FIgure 66. The camera is elevated to-the higher pOSit1011 by means ?f parallel side pieces, thus kept horizontal through palall.cI()gran~ motion. An eyepiece right-angle prism must then be SU\I~.lCd to direct theJmage into the camera. . . Virh these suggestions as to the p~ssible forms which the apparatus IIlt.ght t.aIce,enou&,h data have been gIVen to enable anyone rcasonahl skilled 111 mechaDl~al construction work to plan the particular type of apparatlls best SUIted to his individual requirements. It should I>l' pointed .out, ho.",:,ever, t1;at all designs for use wirh rh mi 'roSCOpt' in tilt' vertical pos!tlon al:c inh rcntly 1110resusceptible to vibrurion: Ihall tl~ost' of the Strict horizontal type, and arc hence less adapt 'd to vcrv
/11

Holes thru Baseboard. cs reqwired

FIG. 64,

Clamps for Locking Support

Blocks

Provision for Extra-Long

Bellows

h power work.

Complete flexibility of' operation calls for a maximum bellows length exceeding that obtainable even with a large-size view camera. When one is forced to use cameras of other types with still shorter bellows, the limitations are still more severe. To this problem there are two solutions worthy of consideration. The first is to employ more than one camera, in tandem; if they are of different sizes, the smaller is placed adjacent to the microscope. It is not.difficult to devise an intesmediate fming,.preferably of wood, to which the back of the first camera and tile front of the second can be attachedin some light-tight manner. This intermediate section should be so designed as to provide a central support and at the same time to be capable of adjustable movemcnt for closing, the entire bellows length to a short projection distance. If two cameras are not available, it is not a difficult matter to make irhcr an entirely new bellows of the required length, or an extension to uch as may be available. Figure 67A shows a simple method of contructing a bellows, Two layers of material are needed: that. to form h . outside can be thin rubberized cloth, such as that used for focussing -lerh, the other, a firm, dark-colored cloth of the texture of fine muslin. The stiffening strips can be made from thin pressed fiber board nhour .0 I 0'" thick, such as that of which filing envelopes are often inadc, The stiffening strips are first glued in position, as shown in Joi me 67 B, to the insideof the outer layer, the latter being laid on a n, It Sllrface for the purpose. After ~hey are thoroughly d~y the inn~r loth is glued over the top of the StrIpS,preferably staggenng the POSIion of overlap of the two layers so there will not be an undue thickI ss at this one place. little ingel~uity is required in bringing the two idl'S together, thus forming the entire structure into a long, re~tangulnr urhc. The outside must he glued in place first; when that IS suffii 1Ir1~' dry to hold togerhl'1', the final portion of the inn~r layer is to lucd down. 0 ant'lllpt should he mad ' to compress Into the nnal

HOMEMADE

EQUIPMENT

CONNECTING THE MICROSCOPE AND CAMERA

.,

." bellows shape until the glue is dry. Then it will be a simple matter to fold the bellows .together. It sh?u~d 'be ~ompressed completely and held together with pressure until It retains the bellows form. As ~hOWI1~ the design provides for a straight bellows. If a tapered bellows ISrequired, the overall layout is as shown in Fizure 68. Each row of st i If ening strips, from the large to the small end~ must be shortened the

99

sing is completed on the g~ound glass, the latter must be removed and the plateholder substituted for it. Then the slide must be withdrawn before the exposure can be made. These operations cannot be performed without subjecting the camera to considerable shaking. If.any camera movement can be transferred to the microscope, the focus is likely to be disturbed. This is especially true when high-power oilimmersion lenses are being employed. Connection is therefore e~fccted between the microscope tube and camera by means of .what IS called a " light trap," a design for which is shown in Figure 69A. ' The two parts of the device can be made of sheet metal by any tinsmith or,

Rownovable
.Support

FlU. 66.

Combined. Horizontal-Vertical

Outfit, with Camera always in Horizontal Position

FIG.

65 Combined Vertical-Horizontal

Outfit

p'l'Op 'I' alll~)lmt over theyreceding hit 'I.

row to provide for the reduction in I~.'. In elthe~' the straight o.r tapered form, the square end strips at '11 -h end provide the box which must mount on the camera front and

turned out of wood on a wood-turning lathe. The latter construction is obviously more bulky, but equally satisfactory. Whichever form is HIed, it is important that there be plenty of clearance all around, so hat there can be no .possibility of touching under any circum-

tunccs,
When the microscope is of the large-tube type, such as shown in UI'C 46, and provided with a funnel adapted for low-power photo, 11I1 'J'ographic lenses, connection for the funnel must be of large size, .I , illustrated in Figure 69B. Under this condition, an additional luprcr can be made to fit 'the large ring and provide the light trap for h . regular tube.. . Simple means should be provided for locking both the front and I ncl of the .arncra in position so that no change iri the focal length can I 'Ul' during the insel'riOl: of the plateholder.

Connecting the Microscope

and Camera

I"

. ~1!hol1gh, as already pointed out, freedom from vibration demands rWJ(liry between the microscope and camera, this must be accom plishcd e'l1tir~ly th~'ough the supporting base and not through any di I' 'ct c()nnectlol~ WIth the tlll~e of the microscope. It is imperative that 1\0 cxrrnn (,>lIS light he perlltltted to enter at this point, yet there 11I11~t ill' IHIphysical COJln ' tion. The reason for this ISthat aft 'I' nil fO('\ls

lOO

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EQUIPME

eo

DENSING LENSES REQUIRED

101

,..
~ k:__
...1-..1

....:L::.::en:..:!oc:::th:,:0::c.'.!..:::clo::::t~h;:25~')\:..o ~m~or-"-e ~th~an"'-_~> 1naximu1?~ bellows extension

B
La P=?i'" v.,'hang of Cloth Full size Detail '1'16' clearance

1~~~~~-_~_~_:~ ~-O'~'~M:::
A =short dimension of bellows B=long C = 3/4 of B. D==V4 of B. C+D=B
FIG,

67, Construction

of Bellows (straight type)'

Control of Light Source The use of a photoflood or concentrated-filament projsction bulb as the light source has already been suggested, Either of these will provide adequate illumination for even the highest magnifications, with color filters inserted, When used at full intensity, however, their lif is limited (especially that of the photoflood lamps) and current conSII IIIp' ion is at a maximum. Experience has shown that the time actuIIJJ~I r '1]IIircd for focussing on the ground glass and exposing the plar I , on the avcrage, only a small fraction of that spent in examining th ' lid, visually and selecting the proper area to photograph. I' 01' Ihis preliminary visual work, the intensity of any light suitable for tal ing thc picture is far in excess of that which the eye can stand. common practice is to employ an absorption disk 'of dark, neutral tint rluss over the eyepiece, or suitable color filters in the path of t he illuminnrion beam to reduce the light to the proper intensity for visual work. There is a much better method which not only adjusts rhc light to till' desired point bur saves .lccrriciry (somewhat) and mar 'I"iaJly pro Ion rs rhc life of the lamp. This is the use of r .sisrnncc in 111(' 1:ll1lpcir cuit, which run he ('\11 III or out as desired. Slidt., wire I"l'Sist:IIH'l'S :11'(' fllt'llish'd hv Ihl' manufacturers of iuicroscopicnl equipment hut :11'('

rather expensive. A good subst,itute can be v,ery cheaply constructed by anyone, with standard electncal parts costlng but a few cer:ts each. Figure 70 shows circuits employing one, two, and three reslStanc.es, with their accompanying switches. The resistances are heater ?l1lts, such as those used in small portable electric heaters of the reflector type. They are equipped with standard Edison bases and hence mount in ordinary porcelain base sockets. The switches are of the regular toggle snap switch type. When a single resistance is used, but one reduced intensity is provided and it may be necessary to modify the winding of the resistance to obtain the best illumination for visual work. Lamps of various wattages could- also be used for resistance, but as they must be of high wattage ratings, they are FIG, 68, Shape of.Bellows more expensive than the resistor units and' Cloth (tapered type) lIIay also be objectionable because of the in" , rcnsity of their glow, unless mounted in a closed box, Wl~h three :esisrance units and three switches, five combinations are possible, which should 'provide ample variation to meet 'any condit~on., . , If photography with an arc lamp as the source of lll?mmatlOn l~necssary, an auxiliary lamp for visual work is often desirable. ThIS can h . a Tow-wattage lamp mounted temporarily in front of the arc lamp md used only for visual work in selecting a ~~itab~e fie~d. ,On~ need not care whether the setup is ideal so far as cntlcallllummatlon ISconirncd, what is most desired is a quick change.-ov~r to the arc lamp -hcn all is ready for transferring the scene of operatlOns to the groundlnss focussing screen. The use of the auxiliary lamp not only proides the necessary reduction in intensity of illumination but saves materially on carbons and current. ondcusing Lenses Required' ",Vhcn horncmade 'const~uction is undertaken 'it is sometimes a diffililt matter to find lenses suitable for the light-source condens,er, as his requires a fairly large lens of short focal length. The desirable undit ion is a cond nscr which will entirely fill the largest aperture of h . SlIhSI:lgt.'cond .ns r, when employin,g the so-called Kohle~ metho:J illumination. ') hrec factors determine whether or not this condi-

.!

102

HOMEMADE

EQUIPMENT

,.

MEANS l?OR CONTROLLING THE EXPOSURE

103

tion is met - the diameter of the condenser, its focal length, and the distance between the light source and the microscope. As the focal length is increased, the diameter must also be increased, and vice versa. Increasing the distance between the light source and the microscope necessitates the use of a larger-diameter condenser or one with a shorter focal length. When the author's method of securing critical illumination,. described on page 192, is employed, the characteristics of the light-source

lens of the substage condenser is not filled, a duplicate second lens can be mounted directly in front of the first, the resulting combination providing a sufficiently short focus. A reading glass is especially serviceable as a secondary centering condenser, located near. the microCombinations

i"Of}dl
I~ ~o~

# 1 closed .# 1 open

:
~

:3
::::9
::3 :3

# t, & #2 closed

:
~ ~

# 1 0" # 2 open. otl: . closed


#1 & #2 open

# 1 closed. It 2 either

:3

#1

&

#2 open

"~" ~

# 1 open. # 2 closed

Adapter for use with Low-po'" wide tube fummei Lens Board of Camel'a

It 2 & # 3 closed. # 1either

#1 & #3 closed. #2 open # 2 closed. It 3 open


#1 clpsed.#2
&

== ==
~
~ ~

-:---m:

::9
:3

::::::====::: -:3
:3

FIG. 6c;-A. Light Trap for Connection between Microscope and Camera

:::9

#3 open

condenser are not so critical since the principal requirement is that it be larger than the back lens of the substage condenser, by a fair margin. This is because parallel, instead of converging rays, are involved, and the distance from the light to the microscope is not a.factbr. In general, for average conditions, a condenser with a minimum diameter of at least three inches and a focus of five inches or less, is ;1 good combination. It can be either a double or plane-convex lens. The compound unit of two plano-convex lenses such as those used ill stereopticons is ideal. These can often be picked up, mounted in a unit cell, from dealers in photographic supplies. Lenses of nlmosr nlly size and focal length can be purchased unmounted, direct from opt icnl manufacturers. '" In an emergency a j-inch reading glass can be used and if rh ' h:lc1
Bausch & Lomb Optical Co., Rochcstcr, N. Y. Spcn '('r Lens Co., IIl1ffillo, N. Y.

# 1. #2. & 1t3 open

FIG. 70.

Control of Illumination

Intensity with Cut-in Resistances

Opt"

Ir
I

ro concentrate and center the beam into the substage condenser, preference should be given here to the longest procurable focal

rh,
1111.\'

for Controlling the Exposure

h . reader 1l1::t'y hav~ noticed that so far no mention has been made locution of <1, shutter for timing exposures. That there is need 11111(,'devil" for a co1llplishin.g this isevident. No light may be 1111\ 'd t Cl rea '!i the plate while the plarcholdcr slide is heing withI \ 11 01' I'l'pl:lc('d,sinc(' sh:tkill~ of rh' camera is incvirnhlc at SlI -h ud 'I' some conditions It Illay suffice to turn Oil nnd off the
111
I

1I0MI';MADJ<:

EQ

IPME

ILLUMINATION

SYSTEM FOR METALLOGRAPHY

105

1:llllp switch, but this is ~ot alway~ wise, as'the time of heating up and ,'oolll1g down, and the dIfference 111 the color value of the light before 11 reaches a stable temperature, may influence the result, A shutter of some SOrt is indicated. It does not follow however that an ,elaborate came~a shutte:- i~ r,equired, One of fairly large di~ .uucrcr ISrather expensive, and If It IS to be accurate in the timin of fractional parts of a second, only those of the highest quality are s~tis-

don rays may enter the vertical illuminator at the side. For a u,niversal outfit a Tvshaped construction with two o~tical benches ~t ~I&,htangles to each other offers a satisfactory solution. Whe? this IS ~mpos,sible because of lack of room, or because rnetallographic work IS contemplated only at rare intervals, a special illumination system can be made to serve. To construct it, however, requires machine-shop facilities and it must be accurately designed and built, Figure 72 shows he nature of this device and the principle of its operation. It is com-

Optic

axis

~fi1

1"
opaq1(.,e

sheet material

Loose fittinu

in

slot

--...,.,
Support

1......j...........::~:.......j+I-~+-++--=--j--E-i-l-l-lt1--".~-H- ~Ji:.t

Beam from .: Lamp Condenser

Block
FIG, 7Z,

Metallographic

Illuminator

for Straight

Horizontal

Outfit

FIG,

7 I.

Shutter Substitute

Short exposures - i.e, under one second - are not idcal for 1111(' roscopical work.
[ncrory.

Irised

III Ill:lll,}' respects a, simple sheet of opaque material, such as hard l,lI1111t:r, nucarta, trans~te board, or metal, .interposed in the path of till' 11 h,l ISequal or supenor to a shutter. EIther a separatc block on IIH' op"c:l1 bench p?rtI~n of the baseboard, provided with a narrow SIOI, 1I -h :IS shown In FIgure 7 r, or a slot in one of the other mount ill 1110 -ks, can bc used to suppOrt the light shield, For use itis mcrclv ",.mnvcd by hand for the desired time interval and then replaced, Shorl e,xposurcs, down to one-quarter of a second, can be made hy Ihi~ means if ncccssary. '
I/Il1'llli'llt1tiO'l1

System for Metal!ography

\Vhl",'e rh .phoromi .rographi outfit is I' quircd ,iril1larily for ntcrul rr':lph,(: work .. t hc h:lseho:lrd should he 1,-sh:!J)(.'( inslt':H(of srrui .hl, 01':1 vert icnl IllICroSCOpl':llld c.uncrn cllIplo)'l'd so th:lt Ih(' illuminn
III

of two units: one mounts in the subs~age rin~ which ?rdinarily upports the condenser, the other mounts m the Side openmg of the rtical illuminator. The former contains a positive lens of fairly long () 'us and two right-angle total reflecting prisms, The extension tube mounted on the vertical illuminator carries a third right-angle prism, I such a device, combined with the reflecting glass of the vertical illuminator, the light makes four right-angle bends, passing around tl-:e mi 'roscope stage in so doing, and is .finally projected Ol~tO~he specIn n in a direction exactly the OppOSIteto that from which It left the Ia-hr source. One advantage of such, a method of illumination is that it ~an be mploycd with any ordinary m.icroscope and ?oes r:ot reqUlre the n to be movable, as in the ordinary metallurgical microscope, ' he recent development of illuminating tubes made from tra?spar.,. nt Lucirc makes possible the substitution of an S-bend of this )11ainl in pla e of the double prisms mounted in the substage,

IO(i Supporting

110 'lEivlADE EQUIPMENT


f'

Table for the Pbotomicrograpbic

Outfit

\ Vhere the baseboard must be placed upon an ordinary table for use, it will be found valuable to employ sponge rubber pads (such as ,ht, well-known kneeling pads) under it to eliminate vibration; three are ordinarily sufficient. \ Vhcn a special table can be constructed for it, the legs should rest UpOIlblocks at least six inches square. Between these and the floor can hl~ i,llstTted sponge rubber pads, which will reduce vibrations to a IIIIIIIIIIUIll. The suggestions given should be adequate to enable anyone to desi '11 and construct just the kind of apparatus most suited to his peculiar needs. From this point on, it is largely a question of learning how to IISl'the outfit. That knowledge, it is hoped, may be derived, in part at lcnst , from other chapters in this book.

Chapter 4 THE TECHNIQUE OF PHOTOMICROGRAPHY

Broadly speaking, the technique of photomicrogr~phy ca?, be vided into two distinct parts - the purely photographl~, mampu,lauve portion of the work, and the operation of the photomicrographic apparatus to produce a magnified image that will correctly portray the appear;ll1ce of the object as seen under the microscope. , The first consists of the darkroom processes - developing t?e negative, making the finished prints, all the knowledge and pracu,?es that are the skills of any good professional or amate,ur photog~apher. The technique with which we are concerned her~ IS la~gely limited to the proper operation of the apparatus, up to and including the exposure of the negative, so as to produce the desired result: This discussion must therefore assume prevlOus knowledge on the part of the photomicrographic tyro, ample to cover th: darkroom technique. ' Only occasionally do the ~wo phases of the ~ubJect overlap to the extent that some mention of strictly photograp~llc matters must he made in connection with the makinz of the negative. Should the beginner not understand such references, because of, ignorance of ,hotographic practice, he should consult Chapter 7, which covers the iasic principles of this part of the work. ' aturally the amateur or professional phot~grapher is alr,eady quipped with knowledge which s?ould ena?le him ,to grasp quickly , rh remaining principles of photomicrographic tec~mque. Shoul~ he, t the same time be familiar with the use of the microscope for VIsual Irposcs, he is s'till farther along, Unfortunately, some, in this ~ateory rashly conclude that they know ,all ~bou~ the comb~ned subjects uul proceed to the actual work - WIth inevitably medlOcre, results, 'h 'y entirely overlook the fact that success ~omes from knowing how I '0 ordinate properly the two parts of their knowledge. Moreover, [rcqucntly happens, they may grossly exaggerate their knowledge ropcr microscopical techniqu:. " here is much more to the taking of good phot~mlcrographs than r .ly allowing the microscopical image to be projected onto a ~en-

?i-

10

108

THE TECHNIQUE OF PHOTOMICROGRAPHY

THE WORKROOM

109

sitiz~d emulsion for the time required to produce a negative of proper density. The fU.rther ~ne advances in photomicrographic experience, the more he realizes this. . .In pr~senting practical information on photomicrography, although It IS ~eSlfable to assum~ on the part ~f the reader a working knowledge of Simple photographic processes, It may nevertheless be of material 1~c1p t~) those taking up ~ combination of the parts of the work for the Iirsr nrnc, to assume their complete ignorance of the general subject, nnd a n.eed for starting at the very bottom. Obviously those advafH.~d photomicrographic experience will be familiar with much rhat fSsaid, and may find only here and there information useful for ifllproving their technique.

JI:

1)'fdhJli'llt1ry Considerations If one decides to take up photomicrography, either as a hobby or commercially in connection with some other line of work, the first consideration must be a study of all the factors involved. The extent of the types of work to be done; the amount of money which can be invested in equipment; the room where the apparatus will be located; the darkroom facilities; the equipment best suited to meet all the conditions - all these must be settled and the apparatus secured that will he hest suited to the job. _
(a) Tbe Workroom SOIllCconditions must be satisfied in the room where the work is to he done, The first is freedom from vibration to as great a degree as po siblc. Vibrations result from so many causes, both external and 1111 'mal, that .some may not be suspected until after the apparatlls is I up. III pnvatc homes, frame structures, etc., they may be caused h p .ople walking on stairs and over floors, closing doors, and the lik '. I h 'S call be classed as internal causes, and are usually subject to con 11'01. more serious internal cause occurs when the floor of the rO()111 wl~ .rc the apparatu.s is set up or the table upon which it rests is nor ~~)ltd:. so rhar even slJght. mo;reme.nr of the operator, whilc ~hc ',POSl11T IS helll!:;' I~lade, results 111 vibration and a cons qucm fui'.i'.y pil'I urc. "'hcn It IS known that such conditions exist, it is usually possible 10 OVCf'(,'OIllC them. . Mml' serious vibration problems arc those .rcarcd cxt crunll ,which 011" unnvoidublc and can only he mitigated by dumpin r the ,'lhotollli

crographic equipment until the vibrations do not reach the ground glass of the camera. The causes of vibrations of this nature include hard windstorms, trucks on near-by streets, trains on building slidings, subway and elevated trains, the operation of steam hammers and other heavy machinery in near-by factories, etc. Here and there the earth's strata are such that vibrations from a heavy forging hammer may travel for a half mile or more in.a particular direction, to the extent that dishes will be vibrated off a shelf or other startling evidences of the forces operating will appear, although in another direction the effect will hardly be noticed a few hundred feet away. For this reason it is a good idea, in setting up a photomicrographic laboratory in a hig plant, to assure freedom from such trouble before going to the expense of equipping the room chosen for the photomicrographic work. Although basements and ground floors, especially when cemented, usually provide the best locations, these are not necessarily the oJ?-ly solutions to the problem of vibration, nor is freedom from vibration the only requisite .. The room should be as free from dust as possible, and also light and airy, yet' capable of being darkened by suitable curtains during the taking of a picture and its preliminary examination <:)11 the ground glass. Freedom from chemical fumes, especially acids, is another important onsideration. Under no circumstances should the apparatus be set up in a room where chemical work is being done, for deterioration will r nder the apparatus worthless within a ve~y short time. Proximity to the darkroom is also a desirable feature. The darkroom itself is a prerequisite, for one cannot expect to car!y on gen~ral I hotomicrographic work without doing his own developing an~ prrntI11g. Of course amateurs, taking up the work as a hobby, can lmproise a darkroom in the kitchen, bathroom, or a closet and can plan to 10 their photographic work in' any convenient room, dismantling and oring the apparatus if necessary when they are th~ough for the eveing. All these conditions, however, should be considered beforehand nd a course of procedure determined accordingly. . .If, as we present the various problems confronting the.photomlcrognphcr, we see~ to take for granted .conditions and eqUlpmer:t far ?eond rhc capaCIty of the humble begmner, he should not c~nslder ~1ImIf ruled out. It is rather merely that, by .the-assumptIon of l?eal ondirions, the entire subject may be cover.ed m ~he manner best s~l1ted () giving rh maximum information. ThIS policy, therefore, WIll be llowcd throughout this chapter.

I I ()

THE

TECHNIQUE

OF PHOTOMICROGRAPHY

SETTING

UP

THE

APPARATUS

I I I

(b) Setting Up the Apparatus


The photomicrographic outfit having been determined upon, purchased, and delivered, the first steps are to get it properly set up and to become thoroughly familiar with each part - its name, function, and mode of operation. To achieve this, it may be necessary to make libcrul IISC of the manufacturer's catalogues and, if any, their instruction Slll'l't Oil the specific outfit. With the simpler equipment there may /1ot he much auxiliary apparatus. 1\ t tuchmcnts such as those for the Leica and Contax cameras require IittI ' preliminary setting up except placing them on the microscope t uh '. \ Vith these, most of the preliminary experimentation will be Iituircd to the manipulation of the light source adopted for use with the ourfir, and the securing of proper illumination. It must not be overlooked that with these simple outfits the same fundamental principles of illumination operate as with the most elaborate equipment. In methods of operation, these minicam outfits are so radically different from even the simplest of the conventional photomicrographi ' cameras that users will do well to master the special techniques cover ing them published by Leitz and Zeiss. A thorough treatise on the Lcica in photomicrography is contained in the chapter on this subject hv Mr. W. Zieler in the Leica Manual. * . \ Vith detailed instructions for setting up and operating these outfits, one is adequately prepared to start work. Later on it will be fourid that much of the matter of this chapter will be useful even in the oprut ion of these small outfits. In the more elaborate outfits, some of the preliminary points to III not ,d arc: The method of extending and shortening the camera bel 1(1\\ si 'ngth; the nature of the light-trap connection between the emu rn and microscope; and the manner in which the camera is moved out of th way to allow visual examination. It should be noted that ill hrin ring the camera light-trap to the microscope, it must always be So locurcd as to exclude completely all room light, and yet there should he IlO physical connection between camera and microscope. The IT:I son for this is to obviate possibility of jarring the microscope out (If focus when the rlateholder is being inserted and the slide withdrawn, Some shaking 0 the camera is bound to occur, but this does no 11:11'111 if the micros 'ope is undisturbed.
I'lIhliNht'd hv M(lr~al1 & Lest r, I,hll' lit """t phllt;, \lIppry denlcrs. 'W York ity, Jf'd cd., prin' $4,()1I, IIIId 11\'1111

Several types of plateholders are in use on photomicrographic cameras. It is necessary to become thoroughly familiar with their operation, especially the manner of loading them, for in many classes of work the loading must be done in absolute darkness. Under most other conditions the faint red or green safelight allowable gives very little light. On the smaller and less expensive cameras the plateholders may be single, and of metal. Some cameras employ standard wooden double holders. The most elaborate holders, used on the large research models of Bausch & Lornb and Zeiss, are of the book type, hinged so as to open in the center. The book type plateholder of the latter is shown in Figure 73, One can practice in daylight, using a suitable

FrG, 73,

Zeiss Booktype

Plate Holder

I iece of glass or an old pegative, loading and unloading until it can be


lone in complete darkness. Especially it should be remembered that he emulsion side always faces the microscope. Thus while the r~gulnr type of holder must have the emulsion up (i.e., toward the outside) h fore the slide is put in place, the book ty.pe holders, whic~ load f~oI? he cent er, must have the emulsion side down. If cut film ISused, It.S inserted in the cut-film holder emulsion side up, and then put into the plutcholder just as if it were a glass plate. With book type hold~rs, no cut-film holder is necessary;. the film is laid in place, emulsion SIde [own, and a glass plate of the same size is laid on top ~f it,. to take t~e hrust of the retaining springs off the film, yet holding It firmly m

place.

..

The operation of the lamp should be studied next. . This ~sespec~ally lcsirable with a hand or automatic feed arc lamp, ItS vanous adjust111 .nrs, centering means, etc. The effect of moving the lamp conI .nscr In and out, or forward and backward on die optical bench, hOIlI(\ be observed. Later 011 we shall see how proper illumination lIIay be secured through manipulation of the condenser. The effect 'of 'Jll'lling and closing iris diaphragms should be noted. If the type of 1 IIIp to he used calls for the use of a cooling cell, this should be filled

-I

IU

THE TECH TIQUE

OF PHOTOMICROGRAPHY

SECURING OPTICAL ALIGNMENT

with di?tilled water or a filtered solution of alum, and put in place on the optical bench at this time. Full~ nine~y per cent of all photomicrographic work is done with rransmirted light, the most notable exceptions are low- and mediumpower work on ?paque objects, usually with oblique-incident light, and metallographic work. The former will be considered later in this chapter, and metallography will be covered in Chapter s. At present we can concentrate wholly upon the problems of transmitted-light photography. (c) SeC1lring Optical Alignment ".'hc p'~il11aryrequisite in this type of work is the securing of correct opt icul alignment of all parts from the center of the light source to the ccnrcr of the camera ground glass. One of the first things which ~hould be done (unl.ess already pr~:)Videdby the camera manufacturer) ISto mark a small diagonal cross 10 the center of the zrourid zlass by mea ns of a soft .lead pencil. This will be at the cros~ing of ~he lines from th.e OppOSIte cor~e.rs. At the same time, if the plate size of the c:.1!1.1e.ra ISlarge, the position of small~r plates, when used in plateholder lots, should be marked. The usual SIzesof plates smaller than 8" x 10" a.('(,' ()1~'x 8t", S"x 7", 4" x S" al:d 3~"x 4~"' The mark for this latter SI/.l',WIll also ~e.rvefor lant~rn s~lde plates, which are 3i" x 4". I wo ~'o~ldIt1onsare pOSSIble10 alIgnment, depending upon whether' Ih' oUlfrt. IS of the vert1~al or h07'lzomal type. Correct alignment is 11111 'h 'asl(,'I't.o secure WIth a honzontal camera and microscope than 11 h I h ' vert ical setup, one good reason why the horizontal camera is 1 I Ihl ' wherever It ca~ be ':ls~d. Understanding the procedure to / 0110 \ xl where. th~ OptIC a~lSISstraight, from end to end, helps ma 11 IIIa vontplishing the alignment of a vertical outfit. I 11 I h '(' ,fore assume that we are setting up a large horizontal out 11 I folio\\' t.h(,(>l~gl~ the P7'0cedure step by step. Obviously one I I Oil tl1l' opllC aXIs ISdefinitely fixed; it is the ccntcr of the camera 11 I luss, This therefore constitutes one end of the axis. The III I I ICJl~ndrlnss should be placed at the distal end of its adjusruhl ' III Ill, I. '" rlll',bellows ~lIl1yextended, cxt the lamp should he I u rh opposlll'md, I h.' extent to which the correct position I I1 ht uun-c run he established on the optic axis at t lux t imc ell' I 1.11(;Il'IO~S. The lamp may not he adjustable; its con I hi 11111 he fr .cd, ulrhou rh Ihl' light itself is adjllslahk; the J 111111vhcn propl'!'l.y alignl'd Illa), hl' indicatl'd hy t h

manufacturer. Or, finally, the position of all parts may be adjustable so that their final positions cannot be set until after the microscope is in place. In this case only an approximation can be made at this time. If it is possible to adjust the light, either by means of the position of its condenser, or by setting to the manufacturer's marks, it should be done at once. At this stage, if the lamp be lighted and its condenser focussed along the optic axis, it should project- a circle of light symmetrically around the cross marks on the focussing glass. If it fails to do so, whatever adjustments are provided must be manipulated until the desired result is obtained. We are then ready to insert the microscope into the system. This is ordinarily. located on a sole-plate, with means for holding it rigidly in place and other means for relocating it in the same position, after it has been removed. As a rule, adjustments are provided for bringing the optical axis of the microscope accurately in line with the axis of the photomicrographic outfit. The microscope is bent over into its horizontal position and the mirror removed. Objective, eyepiece, and substage condenser are omitted, but the iris diaphragm of the substage is allowed to remain. In case a pinhole eyepiece cap is not available, a substitute disk can be made of opaque cardboard which will fit snugly into the' eyepiece tube. This should have a pinhole in the exact center. \ Vith the substage .diaphragrn closed to a pinhole and the pinhole eyepiece cap in place, the position of the microscope on its sole-plate must now be adjusted until light from the light source, passing through its 'on denser, then through the two pinhole, diaphragms of the microscope, registers a small disk of light on the cross lines of the focussing screen. ot until this is accomplished is the alignment perfect. Right here is the heart of ideal photomicrography, and an explanation of much of the mediocre work one often sees produced on the most . lahorate of outfits. . \ Vhen this condition is met, it can be assumed that the quality of the optical parts of the microscope (as to alignment) on all instrumen~s urncd out by reputable manufacturers, is such that final alignment IS assured, except for the minute adjustment which must be made to 1I1i~1\ each objective with the condenser. Some illumination systems require a second condenser in the light rain, ahead of the substage condenser, It is often called (especially by ~ 'iss) :1ccnrcring condenser. It should always be adjustable as to up \I1t!down and sid( to sid .position, when used, but should not be in the J' hr train when mal ing the preliminary alignment, nor should it be mplo cd to corrccr nnv hut vcr minor faults in :1lignment. ,

114

THE TECH

IQUE OF PHOTOMICROGRAPHY

PRELIMINARY

TESTING OF EQUIPMENT

115

(d) Aligm11ent

of

Vertical

Outfits

Alignment of the. parts of a vertical outfit does not differ in theory f rOI11 that of the honzontal type. The complicatior. making this some-

r
1

Optical Axis of Microscope

r-----.
1 _

90 ~ Light Beam at 90 to Microscope Axis

--->""'-'-

--------./

Front Surfaced

Mirror

1
'I
11

The Ideal Condition


with tru.nnion pivots in plane of 'mirror surface.

45

~I 1

r" Double

refection: from. "'",:face

11

Silvered Surface

!lest Condition with Standard Equipment

,:,,,,,s of l,!!ht

Th~ tru~mon

p'vots in axis of Microscope, but below beam. to locate silvered mrface at


Dj

'tntersectton

the t'wo axes.

P?01'.Condi~ionwith Standard Equipment


with. pwots at vnt81'section of axes the refieoted. beam cannot lie in the optic axis.

Poor Design in Some ll1icroscopes The pivots do not lie in the optic am.. of
the

fortunately, several conditions frequently present in the design of the mirror itself complicate the situation and make the problem of alignment one of " cut and try." Then again, the angle between the two axes may be a right angle, or, as is the present common practice, an acute angle, differing with various.equiprnents. The ideal design of mirror - considering the plane side only, the one that should always be used - would have the exact center of the, silvered surface always lie in the optic axis of the microscope, regardless of its angular position. A further desirable feature for photomicrography would be the employment of front surface mirrors . This would not only eliminate the double reflection present in ordinary mirrors but' enable alignment to be accomplished easily by the use of a 'inhole diaphragm cap which could be dropped over the entire mirror. "igure 74 illustrates the ideal condition, which can only be approximated with present apparatus, and also various situations arising in actual service. It is to be hoped that some enterprising manufacturer will design a suitable mirror and mirror arm, with supplemental pinhole diaphragm, along the lines suggested. Another objectionable feature present in ordinary microscope mirrors is the. ease with which they can be moved. Even after considruble time has been spent in securing alignment, accidental contact with the mirror will throw it completely out and all the work must be done over again. 'To obviate this trouble, some manufacturers make a h red mirror, adjustable only by screws. The most practical procedure in aligning a vertical outfit is to emloy an opaque but light-colored card diaphragm over the mirror, t~1e 10Ie being around i" to i" in diameter. The light can be centered on rh, hole, and the mirror adjusted until the light passes through the two pinhole diaphragms of the mic~oscope, to the center of the focussing -rccn. Some workers employ a total reflecting right-angle prism instead of h ' mirror, but this can be done only when the two optic axes are subtnnriall y at right angles.

nticr08cope.

) Preliminary
Frc. 74 Operation
of Mirror with Microscope ill Vertical Position

Testing

of

Equipment

~\:h:~tha.rdel~is that there are ~wo optic axes at an angle to each otlu r, I,h~. POlllt, (Ol.ll~llon to borh IS the 'el~tcr ~)f the Illi<:roscop . mirror, \\ hie h musr he employed to alrcr the till' . .non of t 11(; light I'ays. 11

l Living secured primary optical alignment, the next step is to as mhlc an objective (a IOX 16 mrn.] is ideal for the first experiments), low powcr eyepiece, and the substage condenser on the microscope. It, 'alllcra should he moved out of the way, in the manner provided;

II6

THE TECHNIQUE

OF PHOTOMICROGRAPHY

PRELIMINARY

TESTING OF EQUIPME

Il7

SO that preliminary visual work may be done. A prepared object; preferably a well-stained section, should be placed on the stage and the objective focussed upon it. One-with previous microscope experience will have an advantage here, but let us suppose that the tyro is just starting and knows nothing about the operation of the microscope. With the object in place on the stage and the substage condenser racked up until it nearly touches the slide, the substage diaphragm being opened wide, the object will be brightly illuminated when the lamp is turned on. Care must be exercises! at this point not to look into the eyepiece until means have been: taken to reduce the intensity of the light to a point where it is safe for visual work. Possibly the light can be cut down by means of a rheostat provided for the purpose. If not, color filters or neutral-tint glass can be inserted in the light train, or extremely intense light can be reduced by the use of a dark glass eyepiece cap. When the light is properly adjusted, the eye can be placed over the eyepiece and the objective focussed on the object until the latter is sharp. With a IOX objective, 'this will be when the distance between the object and bottom of the lens is around t to i". For the sake of emphasis we can ask a question here. Are we now ready to move the camera into place, turn the light on to full brilliancy, focus the image on the ground glass, and expose for a picture? Many who have been engaged in taking photomicrographs for a long time mighr answer " Yes," or counter with a " Why not?" But the answer is decidedly "No." We have not yet assured ourselves that the light is 'r irical. If the so-called Kohler system of illumination is to be used, I' is the prcsent common practice with many commercial outfits, the Iirsr st 'P is to move the lamp condenser back and forth until the posi tion is found where the light source, whatever its nature, is sharply illIII 'd on the iris diaphragm of the substage condenser. Thc lan cr '1\11 h ' partially closed to accomplish the focussing, if necessary, or :1 I i ' c of white paper held against it, more readily to discern the imn "1', \ Vhcn the lamp condenser has been designed for Kohler illumiu.i rion, an iris diaphragm is usually mounted in front of it. This is t hI' /h'ld diaphragm. It should be partially closed at this stage, in P" 'P;!I':! rion for the next step, the focussing of the substage condenser. 'J'1l accomplish this, look into the microscope and, after first makillg SIllI' that the object is in accurate focus, move the substage cond .nscr 1I1l1i1 the circle of light, limited in size by rhc partially -los .d field di:lphr:t 'Ill, is also in sharp fOClIS,as well as the ohjccr. The circle of li rht shollld app 'ar evenly illuminated, although it inn not he found to occupy till
Jl

cent er of the field, nor to cover the entire field, which is more or less in darkness around it. Opening and closing the field diaphragm will increase or diminish the area of the field illuminated, thus demonstrating the propriety of its name. It is provided for this one purpose, that the actual part of the object illuminated need not be appreciably more than the size of the circle to be photographed. Any light not actually needed to take the picture should be eliminated, since it serves only to fog the image and lower the quality of the picture. If the microscope is all that it should be for photomicrography, some means will be available for centering the objective to the condenser. The centering may be accomplished either by means of centering screws on the condenser, or by a centering device associated with the objective, such as an objective eh anger. "" Ordinary nosepieces are not centering and hence are not suitable for photomicrography unless attached to the body tube by a centering device of some sort, Assuming some centering means to be present, if the illuminated cir'Ie does not lie in the center of the microscope field (which indicates that the substage condenser and objective are not centered with repect to each other), it should now be brought there by manipulation of the centering screws. That lack of centrality does result from nonIlignmcl1t of the objective and substage condenser can be demonstrated It this point, if desired, after the centering has been accomplished with OIlC objective, by the simple expedient of substituting another objeci C for it. Only by rare chance will the second objective be found o have the illuminated circle lying in the exact center of its field. ~ One other ad justment must be made before the light is perfect. The Il ular aperture of the cone of light from the substage condenser to , h ' object must be made to agree with that of the objective, To do his, remove the eyepiece and look down the microscope tube at the 1 I '1, Icns of. the objective. Now open and close the substage diaIhrugm. The movement of the diaphragm can beclearly seen, as the
The specific type of centering device employed is frequently controlled by the pl' 'Clr microscope stand - i.e., whether it is one equipped with a square or fixed IIIIIHIstag', or a rotating stage. Where the stage is fixed, either the objective or the IIllktl~I'I' 'Il~)' bc provided with ccntering means, but rotating stages, unless equipped Ih their ClWIlccntcring screws, must be associated with a centering device on the , tllh 1lI111', SCIas In bring the ccnrcr of rotation of the stage to the center of the ob11,I' fil'ld, III ot hcr words, there are three parts to be ccntcrcd with respect to each Ih I, lu ilC('Cllllplishrhis result, IWO cent 'ring dcvi cs must be present. These can 1l"III'rilll{ (,Cllldl"lsl'r nnd l'l'IlIl'rilll{ Oliit'I'dvl'; c 'Ill 'ring stage and centering obI 01' ~'I'tlll'I'ill/{ l'WHlt'lIsl'r .uul l't"fllt'rill/{ SIIlRl'. Sometimes the more elaborate nil '"I'PI:CJlidl'd wit l: ,lIllhll'I' ,ldill'IIlII'III',

118

THE TECHNIQUE

OF PHOTOMICROGRAPHY

PRELIMINARY

TESTlNG OF EQUIPMENT

circle of illumination increases and diminishes accordingly. The po'sition of the diaphragm should be set so that its edge can be observed as just coinciding in diameter with the back lens of the objective. The light is now" critical"; we have critical illumination. Because the substag' diaphragm serves to vary the aperture of the system under this St'tup, it is called the aperture diaphragm. Replacing the eyepiece will reveal the best possible resolution of the Ohit'ct, provided that the latter is a suitably stained section and not a slide of diatoms or other unstained object with little refractive index ditl" -rcntiation. The latter are not fitted for making a test of this sort, for reasons which will become apparent as we go on. * ( Jnc single condition in the illumination may be disconcerting to the phot omicrographic novice. The entire field of viewis probably not entirely covered, even with the field diaphragm opened to its fullest cxrc nr. '1 his is not necessarily objectionable in photomicrography, because usually the entire field, not being required in the picture, need not all be covered by the light. An understanding of the reason for the non-coverage will show the logical method of overcoming the trouble. Although moving the condenser farther away from the slide will be found to increase the area of illumination - and this method is often followed by those ignorant of the correct operation of the microscope - it should never be resorted to, as it immediately destroys the critical setup we have taken so much pains to produce. ')'he reason for the small illuminated field lies in the design of 'rh ,lIhstage condenser, which has been provided with a large aperture so I to function with high-power lenses. When these higher-power oh)' 'Iiv 's art' substituted for the IOX, the field will be found to be fully '0\ red. To take care of low-power lenses such as the TOX and lower, III mufncturcrs usually so design the condenser that its top lens can be 11I0 'I. or a supplemental lens used below it. Unscrew the top lens 11 I I' 'P 'at the entire series of operations, from the point of focussing he suhst:lge condenser. The field diaphragm should be closed marcri 1111. for this purpose. The substage condenser will be found to [ocux ut a IlOinr much farther away. The aperture diaphragm opcnin r, whit') probably was only a few millimeters in diameter with .ritirnl li rhting when the top lens was in place, now will be found to require substantial increasing ..~ ftcr the new adjustments arc complctvd,
St't P"K(' 17U

opening the field diaphragm will be found to cover amply the field of the microscope. What has been done is to substitute a longer-focus condenser, which yields.a larger image of the light source (the lamp condenser, in this case). This same principle will be found to operate with still lower objectives, under this same setup, although a point is finally reached where a change to low-power Kohler illumination must be made if it is desired to utilize the entire field-covering capacity of the lenses. The practical application of this system will be taken up later; also, further information will be given on the alternate method of obtaining critical illumination by imaging the light source. The relative advantages and disadvantages of the two systems will also be discussed. For the present, however, let us consider that we have laid the foundation for taking photomicrograph's. Once we have arrived at this stage, the camera can be put into position, the strong light made available, and the image examined on the ground glass. When the image is in focus visually, it will not be in focus on the glass focussing screen, but a slight adjustment of the fine-motion screw will suffice to make the change from one to the other. . The effect of varying the bellowslength on the size of the image hould be noted, and also the variation in image size with various eye,ieces. It will be found that considerable change can be made in ) .llows length without apparent effect on the sharpness of the focus. Ihis may be a surprise to those experienced in ordinary photography. I itferent eyepieces, however, may require considerable alteration in
>I

I 10'1'0111 11 SO'i('1II IIW(II"('lil'1I1 Ml1ndpoinl, when Ih(' lOp I('IIS of IIll' (,Olldt'II~I'1 I I 'I11 0 I"l,d, 1I('('\lnlll' l'ulhlitioll 11Ir ohlllillillK ('I'iti '111li~hl h)' lh(' ~o (,lIlIl'd ",ohlt I

11\thod no longer exist, since the substage condenser diaphragm ceases to lie in the nr focal plane of the condenser. It should be moved farther away to agree with the ulring longer focus, but microscopes -are not provided with means for doing this. , I he effect of this condition is that the substage diaphragm is no longer a true aperture 11 phral;(ll1and a slight vignetting effect is introduced. However, since we are dealing hh objectives of relatively low aperture whenever the necessity of removing the top I 11 is present, the condition is not serious. It can be overcome to some extent, if 11 desires to approximate the ideal condition, by no longer focussing the image of h li!{hl source on the substage diaphragm, but on a card held some distance away, n point substantially the same distance from the back of the condenser lens (conseIII nll~' nearer the light source) as the object is from the front surface, when the 1II!1('IlSCI' is focusscd on the object. This causes the rays to cross more nearly at the nm where the diaphragm should be located. \\'hl'll Ihc author's method of obtaining critical light, as described on page 192, is I !I, Ihis compl i .ation is not a factor, since parallel rays are entering the condenser III IIIl' ,llWl'lurc diaphraglll need not be located at the rear focal plane to secure the IIl'd limiuu io ill IIll' COli' of Iighi. ,\' , 11I1!{l' 11),

IlO

'1'''1':'I'Io:C"IQ I': 010'""OTOMICROGRAPHY

PLATES AND FILMS

FOR PHOTOl'vlICROGRAPHY

121

Ihe fo 'US, but such alteration also can be effected by means of the fin ad j usrrncnt of the microscope, After becoming familiar with the operation of the outfit, using th low-power (lOX) objective, a higher power, say a 20X or 40X, can be su hSIitutcd. Fach step of the process of securing critical illumination should be l'l'pl':1I .d for every objective change, except that when the top lens of Ihl' suhstage condenser is in place and the same slide is used, it is neccs;try only to adjust the aperture of the condenser to that of the obj cIivc, Later on, for actual photographing, a final adjustment of the Iicld dial)hragm should be made in every case to assure that only th ' P:II'I of I lC field actually being photographed is illuminated, o :1ttempt should be made to take a picture until a number of oth 'I' 1II:111l'rS have been settled. Up to this point we can only say that till' prelilllinary steps have been taken; we're off for a good start, l'lates and Films for Pbotomicro graphy A Ithough the present tendency is toward the use of cut film as a sub glass plate for most photographic worl , microscopy is one exception; the majority of photomicrographers st ill prefer plates. One reason for this is that certain brands of plates, 01 spl'l'i:11 value for photomicrographic work, are not furnished in ('\11 tilm. Then again, the advantages of cut film for general work -li i111 n ss, where !)ortability is a factor; small storage space required; possi lulit v of hu I development in a tank, etc. - have no appeal to t luphOfolllil'l'ogr:1pher. He does not need to carry them around to V:l1' 1011 uut sidc jobs; he does all his work adjacent the darkroom, he pr\' I 10 load a single plate at a time (in most cases), expose, and 11':1 ' I lop lr, before taking the next picture; and ample storage span' is
srirurc for the time-honored
I

ultra-violet and infra-red. It is a relatively slow emulsion and possesses a fine grain; both of these characteristics are valuable in photomicrographic work. Moreover, it is fairly contrasty, which is also often a desired feature in photomicrographic work. This plate should be keJ?t on hand at all times, and if one were to standardize on but a single type of plate, in nine cases out of ten this one would give the most all-around service. . (2) The Wratten Panobromatic plate tEastman Kodale Co.). This plate is much faster than the W ratten M plate, requiring only from 30 to 40 per cent the exposure time with tungsten lights, other conditions being equal. It is not, however, for its increased speed that this plate is used in place of the Wratten M .. Its value in special cases lies in Its' greater softness, where the contrast of the M plate is excessive for proper rendition of all the tonal scale of the object. It is often desirab~e to sacrifice the fine grain of the M plate (for the \IV ratten Panchroma~Ic is much coarser in this respect) in order to reduce the contrast unavoidably present in the object. . There is, of course, considerable latitude in the degree of contrast possible with either plate, through the use of the. different d.evel?pers recommended by the plate manufacturers, but 111 general It will be found that the extreme contrast procurable in the W ratten Panchromatic only slightly overlaps the ordinary soft development of the M plate. Both these types of plates are priced the same, but are conside~ihly more expensive than non-color-sensitized, or orthochromat~c plates. This is one reason (but not the only one) why orthochrornatic plates should be used when proper color rendition of the red is not re-

quircd,
(3) Where panchromatic plates are not req~ired (and possibly.not , I 'sired), orthochromatic and non-color-sensitized plates of medium xmrrasr and speed are usually indicated .. It can generally be assumed that the slower the speed, the finer the grain. All plate manufacturers upply plates in this group; each worker sh~uld try O~lt differel~t brands and emulsions for the purpose of selecting one suitable to hIS n .cds, Of tbe Eastman pla res, the Eastman Commercial (ortbocbromnric) and Eastrnan 33 (non-color-sensitized) will be found ideal. Fnsr plates, especially, should be avoi?ed. . . ' . (..f.)' An extreme-contrast plate WIth no color correction IS a?- irnI orrnnr addition to the photomicrographer's stock. Although If t~e ilisolurc limit in contrast is desired, Process plates (not Panchromatic I roccss) will Ill' Found rhc h .sr, a plate of this cia. s, but faster in speed nd snmcwluu kss conI rast ,is rhc l l.muucr Slow plate (I Iarnmcr Dry '.

unll

:1

uilablc,
type of plate will serve for all classe of photolllicrogr:lphil'

0'01\' \ 0"',

On the other hand, the number of kinds which must he 011luuul

to meet ordinary conditions should be kept at a minimum. Of ('()\"'Sl', I hose .\\'ho specialize in but one line of work may often gt'1 alon r ",il h one kind of plate or two, at the most, To cover rhc '1)\ire field (,'iI'I'PI for infra-red work), four different types will he found :1lkqu:If\'. These, \ irh their sp' .ial characr .risri 'S, are as follows: (I) Tb" JYrt/I/(''lI II1 plate (Ra.l'/'lJla'll tc odal. Co.). This \\,:1, dl' vclopcd eSpl't'i:llly for photoruicro rr:lphic work. I1 iS:I pan 'hr()III:l11 plnr " coveriu r :Ill the isihlc Spl'l'II'IIIll,:1I1d sli rhtly hl'. nnd, ill bOlh tlu

122

THE TECHNIQUE

OF PHOTOMICROGRAPHY

MISCELLA,

EOUS EQUIPMENT

FOR THE PHOTOMICROGRAPHIC

TABLE

123

. Pl~te Co.). Thisis a very serviceable plate for photographing objects without calor, and-of low refractive index differentiation. Special branches of photomicrography - i.e., those discussed in Chapter 5 - may require other types of plates, but these will be indicated in connection with the particular work for which they are needed. Regard~ess. of the brand or type of plate being used, it is important that each individual plate be brushed off with a fine camel's-hair brush iJ?mediately before being placed in the holder, as otherwise dust partrc~es may ~e present and cause pinholes. Most plates might not reqUlre brushing, but unfortunately one cannot tell in the darkroom whether or not it is needed, and so, to be on the safe side, always use the brush or some equivalent method to assure freedom from dust. Occasionally a particular box of plates will be found unusually dusty. O~e reason for standardizing on a minimum in the varieties of plates used ISthe fact that plates do not keep indefinitely without some deteri?ration. Fresh plates are to be preferred for best performance. There IS,however, considerabl~ difference in the keeping properties of plates. In g:eneral, panchromatic p~ates do not ke~p as well as non-panchromatic, and fast plates deteriorate more quickly than slow .. Keeping depends largely on the storage conditions. The ideal method of maintaining freshness is to store the reserve supply in a closed metal container in a refrigerator. Even infra-red plates keep well under such conditions. Fortunately the photomicrographer is not ordinarily interested in sUl?ersensitive plates, which are the worst of all from a keeping stand- . pomt. One other thing which can be done to reduce further the stockkcpr on hand is to standardize on one size and consider all others as spccia 1. Where the camera will accommodate large plates, kits can be used and the larger sizes reserved for unusual occasions. Foe commercial work the 5" x 7" size is ideal, but both 4" x 5/1 and 3t" x 4t" pictu res are satisfactory for many purposes and are less expensive. For ordinary photomicrographic work it is not necessary to order specially backed plates to prevent halation. This is not troublesome, even though photomicrographs by transmitted light are taken under the identical condition responsible for so much trouble in this I' 'spt'('t in ordinary photography - that is, photographing dire .tly a rninst IIH' light.

Miscellaneous

Equipment

for the Photomicrographic

Table

A few additional pieces of equipment not recognized as part of the photomicrographic apparatus are nevertheless almost indispensable to the photomicrographer. First on the list is a stage micrometer, for the purpose of determining the magnification of the particular objective, eyepiece, and bellows-length combination being employed. This is a scale ruled in hundredths of a millimeter on a standard 3" x I" glass slide. It is illustrated in Figure 75. An eyepiece micrometer is not an essential for strictly photographic work, but is required for visual determination of magnification. Sometimes it happens that a net ruling or some other scale is required to be photographed superimposed on the object, in FIG. 75 Stage MicJ'OI1Iwhich case a standard micrometer eyepiece Det~r,Enlarged about 'r '11 . . . lameters WIth adjustable eye-lens must be available. The next items on the list are a focussing glass and an ordinary cir cular reading glass. For the former a 6x aplanat il1.a focussi~g rt.:ount is usually employed. The Bausch & Lomb model IS shown 111 l'lgure 76. The focussing feature is essential when it is desired to examine the image through clear glass, as will be explained late.r. For some work, it three-inch reading glass serves better for checking the focus wh .n the ground glass screen is .used. It enab~es a.general survey ?f the entire area to be made and YIelds a compOSIte VIew much supenor t9 that of the higher-power focussing glass, when a thick or wavy section is being photo.graphed. An Eastman Timing Clock (Figure 77) or its equivalent is a desirable adjunct to the outfit. Usually one is dealing in seconds, or split seconds, in c 'posure, and hence a timing device where the seconds are easily read is superior to an ordinary watch. A good stop watch is satisfactory hilt the huger ,10 ,1 dial .an be S' '11 at :l much rrcntcr disrunc '. Other useful .m i 1'11I. 7(" II1111Nrli /'.( 1,01l1b (10l'lIHsill!( (1I1\~sl'S rlt-s include: :l huud mirror for ohscrv

124

THE TECHNIQ

E OF PHOTOMICROGRAPHY

PHOTOMICROGRAPHIC OPTICAL EQUIPME

12

ing the imag.e on the focussing ~lass screen at the same time adjustments are bemg made on the optical bench or where it is not possible . to. s~e the image; an ordinary millirneter ruler; a pair of machinist's diviners for measuring the image on the ground zlass when the light in the room is not sufficient to use the ruler directly; an inexpensive slide rule, fo.r computing exposures; and pnnted record cards for keeping permanent data on every exposure made. A copy of the record card used by the author is shown in Figure 78. Other workers might desire to keep data not . provided for on this card; each FIG ET' Cl I should therefore design a card . 77astrnan Immg oc C to suit his individual needs. Th~y cal: be printed to order by any job printer, 'as there are none available m the market. A combination immersion oil and xylol bottle such as that suppl.ied
PHOTOMICROGRAPH
NF. ATIVE 16U~J CT NO .

by Zeiss (Figure 79) should be at hand, for there is a constant demand for xylol, as well as for the oil, especially when immersion work is being done. Separate bottles can, of course, be used, but as the Zeiss design requires so little room and the base is so broad it is almost impossible to upset it, it is a particularly good one. Finally, a pair of soft camel's-hair (or sable) brushes should be kept on the photomicrographic table, fOFdusting purposes. One should be a flat brush, about i" to I" wide, for dusting the larger condensing lenses, which tend to draw dust, lint, etc., because of electrostatic attraction. The other brush should be a small, round, longhandled artist's pencil brush, for removing lint from the eyepiece, object cover glass, or, when necessary, the back lens of the objective. It is usually necessary to brush the lint from the eye lens of the eyepiece each time it is used, unless the room is free from dust . These few items will be found sufficient for general work, although it is only to be expected that many others will be required occasionally, .FIG. 79. Zeiss Bottle for specific purp.oses. for Immersion Oil Pbotomicrograpbic Optical Equipment

RECORD

DATE ............................... :.. TRANSPARENT .....


,

...........................................................

..............................................................................
MAGNIFICATION OI:lJECTIVE. CONDENSER PLATE REMARKS ................
' , "

...

OPAQUE ....

DIAMETERS EYEPIECE ILLUMINATION FIL TER

DARK FIELD .. CAMERA HOR ........... VERT ..... EXPOSU RE:: .........

.'

The usual microscope employed for visual work is equipped with three objectives, a r6 mm. (IOX), a 4 mm. (40X) , and an oil-immersion 2 mm. (90X). These, supplemented by two, or at the most three, eyepieces - 5 x, r ox, and either a 7. 5x or a r 5 x - constitute the total optical equipment in the vast majority of cases. It is a safe estimate that hundreds of microscopists, considering themselves in the photo microscopic class because they are provided with an outfit for taking pictures, have no other optical facilities than the three-objective, twoeyepiece combination. . . Such a combination is extremely limited, when the entire gamut of magnification range is considered. Even if the amount which can be invcs ed in photomicrographic equipment be limited, if at all possible it should be supplemented by a tax (8 mm.) objective and one of very low power, say a JX or SX. As <1 matter of fact, it would make a better combination to r ph'. rhc .. pH objective hy the 20X and 5X, for the latter will prove more useful ill the lun r run. "'here one l1l:ly huvc n f:lii'l,v complct Iin . of optical equipment, r~le

(j

TilE

TJo:CH

'[QUE OF PHOTOMICROGRAPHY effectively with the lenses listed in the fol-

PHOTOMICROGRAPHIC OPTICALEQUIPMENT tions that arise. The theoretical reason for this was outlined in Chapter I; we can now see how it works out in a practical way. To do this it is desirable to formulate a few rules, which are worth remembering. First, some definitions may be in order. Magnification, it will be evident, is the number of times larger (in lineal dimension) the image (i.e., the photomicrograph) is over the object. That is,

t'llIi~'c range can be covered IO\vlllg table.

'~'his list represents those from the author's own extensive series with \\'1~1~'hat least .95 per cent of his photomicrographs are taken. I h .,approXImate.range given in ~he last column is based upon a maxi11 It 11 11( arncra extension of I 12 centimeters (44 inches) and a minimum
OBJEC'l'IVE

N.A. .07
I I . I I .I1 I I I I

EYEPIECE ApPROXIMATE RANGE


I

t." ( ;Ol"l"tzDagor

11111111111. Planar 11 1111. " 0111111. " 1111. " I 11 o 111111. " 1(1111111. Spencer Photo (IX pochrornar (IX "
IOX
IOX

to 4 diameters

.25
IS

zox
lOX

I 40.\" B

&

" " " " L Fluorite Immer.

(IO.\" A pochrornar Imrner. 1)1) \' " " I 01' " "

.15 .30 .30 .65 .65 1.00 1.05 1.40 1.30

Homal II " I " II " I " II " III " IV " IV " IV " IV

2 " 9 4 " I3 5 " 22 ro " 30 20 " 57 25 " 75 30 " 100 80 " roo 55 " 160 125 " 475 ro5 " 350 250 " 1000 500 " 2000 800 " 2800 1100 " 4500 1500 " 6000

" " " " " " " " " ." " " "
"

Image SIZe . . o b Ject size

= maglll ificati catlOn.

"
"

1111 I 'Ill IIld sl.~d. photographic 16 mm. objective, made by the S ocnccr Lens 11/1I11')'I'd Wit h an irrs diaphragm and designed for use withol;t an oCIII'II: I 111';1'"111111 ""'I'"bll' rurvnrurc of the field, it is a beautiful lens, within its'rang .. . 11 1 'I 1111111\ l'h' 1It\~ !ens o.f the Bausch & Lomb lino. Althou h rated onl , 11\ uld 11 1I1i""lIht'cntIIllage far superior to allY dry 4 111111. g apoc I11'0111111 ) I111111111 ",
" "I
r .

I ilH~h.t,sil.l di~meter. With a lesser bellows cxt nsion 11111 IIlh~;:lt~on IS reduce? in proportion, but should :t. 11 I I 1111 Ih,\I1 Il diameter be satIsfactory, the low range figures I I lid d flllht'l'dmvll\Vard. . I /11 I luu \\ it It slI.t'h series of lenses available, in combirun iOI1 'I 11 It lIel\\ tl'IlSICIIl, considerable overlapping ill Ilia 1ni(i(,:l I11 11 1 I Ills I a vuluahlc asset for III 'Cling numerous condi

The Field diameter is the actual diameter of the illuminated circle seen when looking into a microscope, the field itself designating whatever is included within the circle. If the object were circular and just the exact size to fill the field, field diameter and object size would be identical, and the terms could be used interchangeably. The Projected Image diameter is the diameter of the circular image of the field as it is projected on the focussing screen of the camera. The Camera extension is the distance from the eyepiece (or, to be more specific, the eye-point, just in front of the eyepiece) to the focussing screen (i.e., the position of the plate). Then' our first axiomatic rule is: TiVith a fixed Projected Image diameter, the Field diameter is the same for a given Magnification, regardless of the objective, eyepiece, and Camera extension being employed. This follows from the definition of magnification. One condition confronting the photomicrographer, in every picture taken, is the order of magnitude of the object to be shown. It may be a large object, several millimeters in diameter, or only a minute fraction of a millimeter. The diameter of the field he must include is therefore the very first matter to settle. Each objective will cover only a small portion of the total range from largest to smallest object. corollary of the first rule can be stated thus: With a constant Projected Image diameter, as the Magnification is increased, the Field diatueter decreases. Conversely, as the Field diameter is increased, the ,\Iqgnification decreases. \V orking these rules out in their application to the series of objectives previously tabulated, assuming a fixed Projected Image diameter of 5 inches (i.e., a size to cover a 4" x 5.'1 plate or a 5" circle on a 5" x 7" plate), the Field diameter, in millimeters, at various magnifications; is as follows:

128

THE TECH

IQUE OF PHOTOMICROGRAPHY

PHOTOMICROGRAPHIC

OPTICAL EQUIPMENT

129

EQUIVALENT

MAGNIFICATION I 10 100 200 1000 2000 2500 000 4500 6000

1'0

OF 5 25 25 25 125 125 5 5 25 25 1.25 1.25 1.25 .62 .62 .62 .25 .25 .125 .125 .125 .f25 .062 .062 .062 .062 .05 .05 .042 .042 .028 .028 25 50 509

condition. rays of 5X,


3
1

At
IOX,

A, B, and C are shown the spread of the image-forming


and
I5X

oculars

and the resultant

size of the projected

OIl) ECTIVE

image at the r o-inch distance,

100

6 inch mm. 50 mm. "l mm.

125

~ 111111. (6x) 1/' 111111. (lax) H 111111. (lOX)

.1
l I, ~

111111. (aov)

.1'11111.

(60x) 111111. (,)ox) 111111. (12 v)

umlu-r filltl. "1 lim-ul inch

or
a

.-

-- 2

-20

-200

-- ~
400

--

.021

la

40

100

500

600

800

1200

III this table we see exemplified the two rules enumerated, as well as Ihe fa .r that the sal:le magnification, with its corresponding field di.uucrcr, can be obtamed. by several overlapping objectives, There is here, however, no mention of how the ultimate maznification is obta!lled that is? what part the eyepiece and camer~ extension cont 1'1 IlIIte. r()\,~ard I.t, T~1e last line, giving the number of field diameters lll'CIII'l'lllg In a single inch, will aid in visualizinc the actual size of th Ildd. \ \,I~en ~t is realized that at a magnificatio~ of 6000x the diameter cd thl' enure field shown in a five-inch circle is so minute that 1200 of Ih 'lll l'ollld he placedJn a row in the space of one inch, one obtains an I I led till' cxt rcmc nunuteness of the area portrayed. Should it be deII d 10 photo rruph every part.of a? object one inch long, 1200 fiveI 11I I I IlIl',' lllllSI he taken, which, If placed end to end, would extend I III111dl 'd Il'l'l! I I I 111I "1~ to a consideration of the value of overlapping obj c- . I I III1IC1llll. '1'0 rrnphy, as the combination of eyepieces and camera I 111\ '" IIllpClI'!:lllt part in the result, we must examine the efT 'Cl 1 \ 11'11dilfl'rl'llt power eyepieces are used. 1111 I\IIt "llioll IIf the projected image at a distance of 10 inchcx I vc pO~llt is idenr i 'al with. that of .the visual image. Cons I 11 I 10 inch cnmcra tension the Image produced by a IO.\' 11h dOll"ll' that of:l 5X ocular with Ill' same ohjc 'live. 'I his 111111III .1"ClIII ill ollly one way: the :1ngl' of the higher power 11111 I hl' rrv.ttcr t hun that of the lower. In orh .r words, Ihl' lIIIuhlUlr I III pi oj , '( .d iiuu re is increased. Fi rurc Ho illustrates t hi ..

.'

15x ocular

FIG. 80.

Effect of Eyepiece on Magnification

'From this diagram it might be assumed that measurement.of the projected circle on the ground glass. would sho,,-: the sat~e rela~Ive proporrion ill size, but this is not the case. The diagram IS predicated upon the field diameter's being allowed to remain the same. A~tually th.e lil'ld diameter is .ircumscribcd by the diaphragm in the eyepIece .. This li:lphn~glll restricts the diameter of the field to a greater extent 111the

THE TECHNIQUE

OF PHOTOMICROGRAPHY

PHOTOMICROGRAPHIC

OPTICAL EQUIPME

TT

I3

higher~power .eyepieces, ?nd hence there is not a proportionate incre.nse 111. the s~ze.of the .cucle .. Measl~rement of the diameter of any O/JIL'ct ly1l1g within the circle, WIth various power oculars, will demonSI rate that the angle does increase proportionately with the magnifying power of the ocular. _

10x objective a d 70x eyepiece

extent of variation in the size of the final picture required, with other characteristics constant, ranges from the covering of an ~" x ro" plate, to a single frame of r6 mm. motion-picture film. Only with a complete set of objectives and eyepieces can such. wide range b~ accommodated. The following table will be of value m supplementing those already given. It gives both the magnification (in pare?th~ses) and the .field diameter in millimerers of the commonly used objectives and eyepIeces at a projected linage distance of 10 inches. The field diameter is con:puted on a s-inch diameter for the image, alt~lOugh the sx oC~.1lar"v.Ill not usually yield a circle larger tha.n about 3~ mche~ at ~he r o-inch dISranee. For other sizes of image CIrcles (X H1ch.es 111dIametel) at the same projection distance, to find the actual field included, multiply the gi~en field diameter by X. Should the same field diameter be, desired, 5 but a different size image required, the magnification will be the factor affected through change in the projection distance. ~o fi~1d tl~e mag. projection distance nification in this case, multiply the figure gIven by .
r,

-----------20'~"-r--------

10

OnJ EeTIVE

OCULAR

5X (25x) 5mm.
(5) 25 (lOO) 1.25 (200) .625 (300) .42 .28 (45) (6(0) .21

75X (37x) 333


(75) (15) (300) (45) (675) (900) 1.67 .83 .42 .28 .185 14

10X

r25x
(62X)

15X
(75) (15) (300) (600) (900) (1350) (1800) 1.67 .83 .42 .21 .14 .11 .07

20X
(roe) (200) (400) (800) (1200) (1800) (2400) 1.25 .62 3r .16 1 7 5

5x objective and 7 0x eueniec

5X
10X 'lox

(5Ox) 25
(roo) (200) (400) (600) (900) (1200) 1.25 .625 3r
.'21

Fill.

HI.

Illustrating Create.r PI~te Covering Capacity of Lower-Power Same I'.yeplcce, for the Same Magnification

Objective

and

40x 60x
()OX

l'lOX

.14 .10

(r 25) (250) (500) (75) (1125) (1500)

2.0 1.0 5 .25 .167 .111 .083

"'lIt'll we come to superimpose on this eyepiece effect a variation in the C:IIIlt'J":1 length, the result indicates the flexibility possible in ob ainill r a (It-sired df ccr.
,'j rll~"l'H I, sl.1O\\"s Ihe .e(fc. 't upon the image of the same eyepiece

. From what has been given above we are in a position to add a .few . n;ore simple rules. These are: With a .giv:n objective and eyepiece,

wh 11

I1 d \\ 1111 dllll:r~'lIt ~)bJtT,tl,ves an.t! .varying the camera length to obruin till' .lIl1l'III:1 rllrllC:IfIOIl, I he stnklng di(ference between the two con dlllllll j. t ~lt' il1~"'l':lsl'~1sizt' of Ihe projected image diameter for Ihe: 111111111 11111('11111111, \\'11hi ht' lower-power ohjccriv '. . 1111 \\ l'(' rluu l'''.l'l1 ~Vhl'l1.similar Illagni(icalions are producihlc I tI d,lll'J('11f IlbJl'('tlvl'S, 11,dots ~l()t IIlt:an (hlplicalion.of eqllip 1111111J( I .t1\\.IP'Slll1ll'Val"lahklllthl'(III:rIITStrlllhallsofv:t1tll' 11I1till" '0111111111111 arisill r ill phounuicr rraph , The

umgnificauon varies directly as the projection distance from the eyepoint (Ramsden circle) , .. . .
Based upon this fact, a .simple me~hod of.graph111g magnification possible. This meth?d will- be desc.nbe~ a little later. '.' IS

J.flith a given eyepiece and magnification; the actual field ~za11:eterts ill'/.!ers~'yproportional to the ini,t-ialmagnification of the obJectzv~. IVith a giuen objective and fixed projection distance, the magnificaI ion mtd proiected huag diameter (7tSually) are increased by the use of bigbcr po er eyepieces. . . .
Rt r:\I:dkss of
Ihe t ypt'

of camera

employed

- whether

rrumcam,

'32

THE

TECHNIQUE

OF PHOTOMICROGRAPHY

MAGNIFICATION

ITS PRACTICAL

DETERMIr

ATIDl

'33

fixed projection dist~nc~ camera, motion-picture camera, or large research model - application of these rules will enable one to determine how to obtain the maximum variation with the equipment available. \ Vhcn the special flat fie.ld eyepieces of t~e Homal type (see pages Ho :lJ.,d r 65) are emplo):'ed l~stead of the ordmary series, the flexibility ohl uinablc by subst1~utmg dIfferen~ eyepieces is materially limited, so Ihat ~hcse are not so Ideal for very simple cameras. Their use is largely con tined to the elaborate research models. A lso, there is not the fle~ibility of the compound microscope where lo\\" pow 'J"lenses are required, as these use no eyepiece. Hence the I p het "',een, the focall~ngths supplied are graded much closer than , 1~lill' ohJec~lves, Starting with a 32 mm, (sx) in the latter, the steps 11, 11\ the rat1~: I, ~, t, wh~le a series of five Planars, starting \\'11 It I 111111" ISavailable m the ratio: r t t t t, In this way suitable llc ihili: Y is provided, ' , , ,

~,n,

(H)

IIJllglli/ict1tiOl1

-Its

Practical Determination

It is il1lport~nt to know and record the magnification employed for e~~ry ph()tolm~rograph taken., In setting up for the picture, two conditions a,re possIbl~, T.he first ISthat ~he obje~t, with a suitable margin :1~ollnd It, IS requ,Ired JUs~to fill an Image CIrcle of a predetermined dlalll,eter. The diameter ISusually set by the size of plate or film de~cI"I1\lnedupon" The exact magnification necessary to accomplish this I~ mconscquential, except that after the picture is taken, it is then de siruhle to !mow the magnification actually used, both for the purposc of recording on the back of the finished prints and for permanenl record.
',rhe alt~rnate c~ndition is to predetermine the magnification most satlsf:!('t(),l'dy showing' the subject, and then establish the combination of ohJt'('tl~e, .ycpiccc, and.bellows length giving the desired result, In r 'm:ml rllls,latter l1I~thod ISused for subjects not possessing a definite (~IIIII1\l' 01'(,II'CIIIIIS(.'l'Ih .d :ll:e,a ,extended surfaces, tissues, smears, pal' t iclcs, l'll', and the lllagl1lfl .anons employed arc round numbers, hi till' Iunuer l'ast' rhe objects nr entities whi h must he shown entire \ I III IIt ' hi rhl'SI possihle llIagnificarion for rhe sal e of maximum d(, ' r Ill.

the measured bellows extension, This would give an approximation only, For the measurement of magnification, a stage micrometer is a necessity, This is in effect a minute ruler, on glass, The scale is usually 2 millimeters long and subdivided into ,0 I mm, By placing the stage micrometer (Figure 75) as an object on the microscope stage and projecting its image on the ground glass focussing screen, the. amo.unt each division of the scale is enlarged represents the magnification. Two other inexpensive pieces of equipment are required, One is a ruler, graduated in millimeters, and the other a simple pair of dividers (a draftsman's compass with points.. or machinist's dividers). The latter is useful in spanning the scale divisions as seen on the ground glass and transferring them to the ruler, in lieu of superimposing the ruler directly on the image of the scale. . ' Instead of measuring the image of a single division, it is better to include as many as possible, to minimize error in the reading, F or .i~stance, if 10 divisions on the image (each representing, / I 00 of a millimeter) measure 2 centimeters (20 mm.) across, one division would equal 1/10 of 2 cm., or 2 millimeters. To enlarge '/' 00 of a millimeter until it equals
'2'

mm, requires a magnification of

_/2 I 100

200X.

1 IIn 11111 I r hl'l'l'fol'l' hl' nvuilalilc in horh instances to d .rcrurin 'till'


111/1

1III0l\i i,l i, It,ll I sllflil'il'l\l 10 dt'lll'lHI ~'1lO~1 l'OlllPIII:lti(~IIS\):lsl'Il 1111 111ft UIIClI\'1\ raved Oil I I' objective and e, Cpl x'c, alld

Many workers place the scale in position each time a picture is taken, to obtain the maznification. This requires considerable time and is unnecessary. It i~ a simple matter to determine the magnification for each combination of objective and eyepiece once for all, and then plot the results on a piece of graph paper for ready reference. Figure 82" shows such a graph as it appears when ready: for use.. ., According to the rule already stared - that with any gIVen objective and eyepiece, the magnification varies directly as ~he dista?ce ~f ~he projected image from the eyepoint - the niagnification relationship ISa straight line, .One need only establish two points on the graph and ~onnccr them by a straight line to indicate the magnification at ~ny proJection distance, Though this also holds good for extr~polatlOn on b.oth sidesof the points determined, it is preferable to establish the two pomts ar as near the limiting positions as possible, ') he camera extension positions, indicated at the bottom of the graph, will he 'identical in every graph, for the same c~mer~, regardless ~f the I 'I\S combination, but the spacing of the magnification values will reItllil'l' 1'l'l'OI11IHII ing for cac,h setup. ,The subdivisions ~or the mag.nificaion should he such Ihat intermediate values arc easily determined -

THE TECHNIQUE

OF PHOTO lICROGRAPHY

FILTERS' AND THEIR

CHARACTERISTICS

135

1hat is, they should represent steps easily divisible into the base figures, I, 10, 100, or rooo, as the case may be. In the graph shown each group of five lines represents 50X of magnification; the lines, therefore, are in SIt'pS of TOX. The shortest projection dis-: 1100 tance possible, with bellowstype cameras, is the distance to at the plate when the bellows is completely collapsed, and so 4110 the graphing of the magnification never need go below this 110 point. , Workers using fixed-focus , ,.wu cameras need not prepare separate graphs of magnifications, /l0 for a single table can show every possible combination. Reliance should not be placed on 200 manufacturers' figures, which are approximate only; even 60 r with roll-film minicams the exlOO act magnification should b 40 60 00 70 80 90 100 710 determined for every combina0""(,,,,, J,'a;tcn~on Vn Centumeters tion. This can be accom1111., HI. Typical ,raph of Magnifications plished by removing the earnera back and laying a piece 0 111I 1'01llHI glass, ground side down, over the film guides. The stage 1111 'Will 'I er can be projected onto this and measurements made in the Id, crihcd [or larger cameras.
wi: h
(;ill'll

Objective

and Ocular

Fill

If! 11IId

'l heir Characteristics

OIllP '!l'IWC in photomicrography requires some knowledge of rhe nil Ill" of li rill, especially visible light. Light is the medium employed COl" producin r the image on the sensitized plate or film, and rhcr ,fon' Oil ' IlIl1S1 Iwo\\' how to control it. That portion of light which ',\11IH' pi'l 'd lip by the human eye is compos d of vibratory ellergy P()SSl'SS 111 r wuvc lcn rrhs between about 400 to 700 1I1J..L in length, These wuvc lcn n hs of li rht l'IIlT r , if ullowcd 10 fall Oil lilt' t'yl' in n. narrow hnnd (or rl'Ollps) produce the sensation known as col or. The shurtc I

waves are violet, the longest, red. Between these extremes lie all the colors constituting the spectrum (e.g., the rainbow). Only when all the rays composing the visible spectrum are present is the sensation experienced that is known as white light, Taking out any rays gives some kind of a color effect to the remaining light; the exact color depends, of course, on which wave lengths have been eliminated. ' Transparent substances are those which allow light to pass through them. When every wave length can pass through with equal ease, the light transmitted appears the same as the light entering the substance. We then say the substance is colorless. Many transparent substances, however, will allow only certain wave lengths to pass through; the others, either wholly or in part, are absorbed by the substance and so never get through to the other side. The light passing through is said to be transmitted; that not transmitted is absorbed. When a piece of glass appears red, it is because this is the only color transmitted; all the rest of the rays constituting white light are absorbed. Similarly, a blue glass transmits only the blue rays. Hence if we pass white light first through a red glass and then attempt to pass the transmitte-d red rays through a blue glass we find that the blue glass nbsorbs all the red rays and no light whatever gets through. On the other hand, replacing the blue glass with a second red glass allows the red rays to pass through unhindered. This transmission and abso~ption principle is employed extensively in photomicrography to obtain the d 'sired image of a colored object. Since a large proportion of objects which must be studied micro= -opically are colorless, they require artificial coloring (staining) to make them visible. Even blood, which we are accustomed to consider .1 rich red, when spread out in a single layer of corpuscles, on a slid~, is n nrly colorless and must be stained before it can be properly studied, 'raining technique has been developed to such a' high state of perf~c~ ion, through the reaction of dyes with various tissues, that several diff I' .nr colors ate possible on one section of tissue. \ "hen these are to be photographed ~nd interprete? in blac~{ and hire, the various colors present reqU1r~ rend~nng 111, so~e .mter111 xliurc half-tone which conveys-to the mind an 1mpresslOn similar to 11 'l'olilrs in the object itself. For this reason color filters find exteni l' 1I1ll' in photomicrography. ,. . One [actor in both naturally and artificially colo~ed objects, ~s well in .olur filters, of great value in s curing the desired results, 1Sthat

136

THE

TECHNIQUE

OF PHOTOMICROGRAPHY

FILTERS

Ai"D THEIR

CHARACTERISTICS

137

the line between transmission and absorption is seldom sharp. -From substantially perfect transmission there is often a grad~al inc~ease i?absorption until the maximum is reached. Moreover, this maximum IS seldom 100 per cent absorption; itmay vary anywhere between a low value and 100 per cent. These conditions will be evident from a study CIf1 he characteristics of color filters as illustrated by graphic means. The filters employed in photomicrography are of three kinds: liquid, dvcd gl'latin, and glass. All three function in the same ~v~y; the ad\ ,1111 a res and disadvantages of each are largely the determining factors 1111 hl' extent of their use. Liquid filters are solutions of colored chemI 'Ill salts in water. The solutions are inexpensive but require a suitable I.lsScell for containing them. Such cells are expensive and if a corr~pll'll' set of solutions is kept on hand for immediate change, a C~llIS needed for each color. This is as costly as the purchase of gelatl11 or rillS filters. Some workers employ the cooling cell as the container for the filter solutions, but this involves a lot of work in changing from on' color to another. .. One advantage of solutions is the ease of altering the concentration of the chemical compound and thus securing an. immense num.ber of ariarions in color. The absorption of the solution changes WIth rh . concentration. A serious objection to the use of .liquid filters is ~hc difliculty of maintaining uniform standard solutions. Evaporat~oll alters the concentration and sometimes chemical changes occur which afTect thc calor. Unless a color comparator is available to keep check . on the densities, exposure times will be affected. I >yed gelatin filters are produced in the form of thin sheets, made hv the Fastrnan Kodak Co., under the name Wratten filters. They arc }'rClcurablc either ~1l1mounted,. in the thin sheet form, or mounted ill 1mls.uu, between pIeces of optical glass. The latter are prefcrable., :IS rh - unuiountcd sheets, though much cheaper, must be protected agalllst s .rnrchcs and finger marks by glass plates. / set of nine filters, in a compact box, is furnished especially (Of microscopical work. It is known as the M set. The code letters :u\(1 colors in this set arc as follows:
!\ Filter

7
8 9

F G H K-2

" " "


"

Dark Red Orange


Deep Blue Pale Yellow

Brighr Red
Green

B 3
of C
J)

" " "

Blue Vioict Purple


I,iglll Ornn
J"

I':

"

Ih'd

The time factors on these filters, for tungsten and arc lamps and for different types of plates, as furnished by the Eastman Kodak Co., are given in the table on page 138. Filter D (purple) is not used alone, but only in combination with one of the others, and hence does not have a factor given for it. Glass filters, in anything like a complete series, are made only by Schott & Gen, the Zeiss optical glass plant, at J ena. They have optically ground surfaces and can be obtained with practically any desired characteristics. It is possible to obtain an equivalent set in glass, to correspond with the Eastman W ratten NI set. As to the relative advantages of the dyed gela~il1and glass filters, both have certain things in, their favor and also some objectionable characteristics. The glass filters are much more expensive and more easily broken, especially the very thin ones (I" mrn.): On the other hand, they are absolutely permanent, while some of the dyed gelatin filters will deteriorate rapidly under intense light. Excessive heat is hard on the balsam-mounted gelatin filters, causing the filter film to soften and the balsam to run, but if heat of the same intensity be applied unevenly to the glass filters (as is nearly always the case with a focussed beam), the danger of breakage is great. .On the whole, the final determining factor in deciding which type to purchase will probably be the relative cost. As to the exact correspondence of the glass filters to the W ratten M , set, it must be recognized that the eye, unless specially trained, is unable to appreciate subtle differences by visual examination alone. When there is an appreciable difference in shade of color, the eye can detect it, hut not the degree to which the transmission characteristics of filters lIlay vary. To determine this accurately, recording spectrophotorneters are available, which automatically plot the transmission curve of any given filter. As there is very little comparative data available, covering the various filters used for microscopical work, the author, with the collaboration of the l Iarrnon Color Works, has worked out a representative selection of filters -li\[uid, gclatin, and glass - upon which graphs have be.: 'n made on a Ccn .ral Elccrric R cording Photometer. These

..

138

THE TECHNIQUE
I

OF PHOTOMICROGRAPHY

FILTERS AND THEIR

CHARACTERISTICS

139

~g ~~ ~~ g~ ~ ~~ ~~
19 N
0'\0

....

r---O"I
V)

\000

N~ ~o ~r--....,....C"'lq.~_
V) V)

V"IO

VI

V'\

C"}'

M ~

V'\ \C)

"S

C')"

("") ~

51>+51 H+9 SH5C HI6v

a+o
H+v:)

~:2 :::;~g:;:::; 5;8 88 ~5; 85; -N----NMv--....,o -N~I go CV) \')0 00 00 00 00 \0\.0 ~~ "1\0
_00 ~~ \O\,() V)\,Q

00
00 '"

"'n

00

~?2

S~ ~~
....

"''''
0'"

g~ ~~ ~~ ~~
Oc> 0", <" .

H I L~ 11+ D Ii'I 85 l l+ H
~)+ H

~g ~~ ~~ g~ ~~ ~~ ~~ ~g~::! ~~ ~Q 8~ ~~ 8?? --(""'1\['.


-("I -C"I

"'-

I1 I K~ \ ,I KS I1 I

V"O
-("I

00("1
-

OON

,",,0 cv-, --c")~r--.

f"-..C"l
-("IV)

00

"'''' - <"

':11 H 0+\1

g~~R~R~g~~

st

V')

'"
,,...

s (;X

v;
.,....IN -N":N
V)~

V)

V')

('It""')

N'<t"

Nr-~
V')

r")"=t'

e ....
0

9
I 11

1)[

v:.v:
--

v;
--

V)

V"\V'\

--

-("I

C"'I("")-N

NN

ZX
IX

-o

00

~ ~

5~ I-I
51

~ 1:1
Q)

'"' Q) .... ::=

9 .!l
~oo 00
("I

"''''
0'\0
(".

6Z

\0\0

__

00

('.

CC: :1
61' I>;)

v-:
1""'1""")

v:
.q\,Q c")\C)

--r r-,

..;f" 0

,f

ov r-,

".

S.)
11
00 '"

HI

("1 .

,..I'l ~ t1 ~

'"

-t- ~

~ ~

graphs are shown in Figures 83 to 97. To accompany these, corresponding curves were run on several of the common stains employed in histological work. These are shown in Figures 98 to 105. Though the curves of the stains represent definite concentrations of the dye solutions, these cannot be taken as the absolute equivalent of sections stained with these dyes. In many cases the dyes react with the tissues to produce a modified color. This is .especially true in the case of hematoxylin, where the reaction results in a visual variation extending all the way from a reddish purple to a rich blue, although no blue exists in the stain. In spite of these variations, the curves of the dye solutions should prove helpful in determining which filter will be most likely to give a desired result. The general law to/follow in the selection of a filter is that one with a transmission curve similar to that of the color of the object will give the greatest amount of light through the object, with, consequently, the least amount of contrast and the maximum detail. Such a filter is known as a supplementary filter. ' For maximum contrast a filter which absorbs the light the object transmits will give the best results. A filter of this type is called a complementary filter. Where an object has been double stained - e.g., blue and red, as in the case of hematoxylin and eosin - a filter intermediate between the two colors will give the best results. This should be a green filter for maximum contrast, or an orange filter for diminished contrast. Filters are useful, not only in connection with stained objects, but for providing the sharpest image with achromatic objectives when photographing such subjects as diatoms, for maximum resolution. sually, for this class of work, a green screen gives superior results. -This is because the spherical. and chromatic aberrations are corrected to the highest degree in the green region, so that if all light other than green be cut Out, an achromatic objective will yield a picture almost the equal of an apochromat, under the same condition of illumination. The superiority of the apochromat is demonstrated in its ability to render Just as good results in the blue-violet region, where the resolution is hi her because of the shorter wave length. Filr .rs can also be used to reduce the intensity of a strong light when mal ing a preliminary visual study of an object preparatory to projecting the image on the ground glass. The special filter, the curve of which is given in r igurc 97, is particularly valuable for use in connection with an an: lamp.

THE TECHNIQUE
700

OF PHOTOMICROGRAPHY
10 0 100

FILTERS

AND THEIR

CHARACTERISTICS
100

90

C( pp,er S~l h te ~ol ti n -.....

90

80

.-

'\'\

I/V'V
80

90

Jen

Glas~.....,/1/ I- I-"

90 . 80

1'\

1\ 1\

\
10%

1\ 1\ 1\
110 \ Amm. (>niacal \ C?P11er fS'ul,Phat~ 40

1\

2%

11 I 1 1 / 1 11
,I
0

80

"
A,
}I

as~m<n Wr tten

70

,~\
\

'lte?' B Filte?is

1\ \
\

\ \ \
\
,01

Pot (lS i~m Chr mat S lution

Hho
CuS

270 Cc 45 grns,

N{14 OF{ 30 CC

"" 30
20

1\ \
\

1/ \
\
20

1
,

'-1% So,

\
\ \

1\
1\ \

IIf 1\\ '\ rt


20 20 10 ) Wavelength in Millimicron.

70

\
\
'\

/ 1/ 11/

l\
Eastma Wrapten

10

I"

11
11

/
I

10

1\\ lA'" Jena


,JI\

Glass

jJ

I\:
f'"

400204060805002040608060020406080 Wavelength in MiUi1nic1'ons

700 400204060805002040608060020406080700 Wavelength in Millimicrons

~W~OOOO~W~OOOO~W~OOOO~ FIG. 87

~W~OOOO~W~OOOO~W~OOOO~
Wavelength in Millimicron.

Il

FIG,88
100

700

00
80

Data sitn

Dichromate ~ olution

I I I I

100 90

700

I1I ~

80

,-,
I

":"1,

90

90

80

80

1
D F,ilter

10 -

1/
I

00

,%
/10
1-

Pi
'n

%
- 1-"

01,

Ho'

7'i'

Ac d,
I \.

I--J ~

Glas.

I~

40 11-

-I1- I~-

C Filters

1'\
.

30
20 1-

\'
\""
East'l'nan

-20 1-

'-1-

1- -1-

10

--

VIJI)
FII;. H

- ---- J"'U.".,

70 -

.- J

=
20 A

Wr tten-

f- t-""

.I

20 lass 1.0

70

'I

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THE TECH

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FILTERS AND THEIR CHARACTERISTICS


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100

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I45

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THE TECHNIQUE

OF PHOTOMTCROGRAPHY

TI-IE LIGHT SOURCE

I47

, J\notl~er t,Ype o~ filter, especial,ly valuabl~ when an arc lamp is used III combination with a filter passmg ultra-violet rays, is one that will eliminate the objectionable ultra-violet. That is, the two filters should Ill' used in combination. This is necessary when objectives are not well corrected in the ultra\ ioll'l I'C"ion, Plates sensitive to these short waves record an image in Ih " lilt 1':1vi(~lct ,:hich is not ,apparent visuall~ on the ground glass. 1111 ult rn VIOletImage, superimposed on the visual image, lowers the '111,11 il \' of Ihe picture. I hOlolllinographers who possess a microspectroscopic eyepiece, a h uul sp 'cl roscope or more elaborate model, can make cood use of it III IIIdr.il~g the transmissio~ bands of unusual filt~r combi~ations, and I IIIIS. I Ill' knowledge gamed by such study aids materially in obruinin r a conception of the relation between the visual appearance of l'olol' 'd oh jeers and their transmission characteristics. One should not be content with his results until he has completely mnsr 'red the use of filters. One rule that is particularly worth remembl'rillg is, never use a more contrasty filter than is absolutely necessary 10 produce a satisfactory picture. What is wanted primarily is detail, 1101contrast. .
/,(,~'i/l.'l, tbe

Picture

l ndcr this heading we can group the necessary steps in the produ '- ' Iloll of it phoromicrograph, from the time an object is placed upon the , 1It1III 'lIpl' SI:Ire until the negative is ready for development. III f I 'Iill'Sha vc ;1 hearing on the actual impressing of the image 011 I 1111d pl.\ll" Fach of these is so tied up in some manner with t he I I 11I h v ',111 be conceived of as parts of a whole, rather than :IS I 111111 'I fill' -onsidcration, The term "exposure," per se" IT I I 1111 l of t imc during which the light is building up a latent I Ih 111111 'd plate. This period of time is influenced bv so I I11 Ihili il lI1:1yb ' of any duration from a small fractio;, of I I1 III '\' .rul hours. It therefore becomes n c'ssary 10 "ndtll'lIllIl 1111,h 1111I,lIl1ll'!' each factor affects the time clement.
,m /II/lII('lIcillJ!, 11.1(' Tnue

time during which it acts on a sensitized ,emulsion ~re ~quiv~l~nt factors in effecting a definite amount of darkenmg. For instance, If ~thas bee? determined empirically that full blackening of a plate of gIven sensitivity is produced by roo units of light ~ner~y, this result could b~ obtained by any number of different combll1a,tlOns. ,As an ex~mple.' l~the light intensity amounted to t oo units, the time unit f?r wh~ch this l~ght must act would be r unit; 50 light units.would reqmre tW_lcethe tllT~e, i.e., 2 units; while a faint light intensity of r would reql1lre 100 units of time. This is known as the reciprocity lan. It-has been proved that this law does not always work; it has a substantially straight line function over a considerable range, but beyond this range the line becomes curved. Thi~ is called the failur~ of the reciprocity law. To illustrate, if we continue the case mentioned, r /r 0 of a light unit acting for 1000 units of time should produc~ the full blackening. When it is tried out experimentally, however, It m~y be found that only partial blackening has been effected: Indeed, If we lower the intensity of the light far enough, no blackening whatever results regardlessof the extent of the time it might be allowed to act. We have then passed the threshold value of the ligh~ for a given J?late. Fortunately, most of the work of the photomicrographer lies on the straizht-Iine portion of the reciprocity law curve, and hence he c.an figu;e his exposure times on a scientific basi~. Only when a ve:y faint light source is involved should compensation be made for failure .of the reciprocity law, by increasing the time over what would otherwise be required. . _ ,_' ,'... , In microscopy, the mtensity of.the light sour~e ISt-'le mtensity of tl:<1 original light after it has been subjected to modification by all the vanous factors which alter its intensity. Most of these factors are of such character that their effect is mathematically cal~ulable after a basic ex'posure time is once determined. This simplifies our problem somewhat. The micrographer should know just what to expect from each variable. To this end, let us analyze the part they play. (1),

The Light Source

oj' Exposure

1111111'1' '111111110111 :lCt' 'pit'" as roverniu t IIll' npOSlll'l' 111111' 1Ir! is rluu the intcusit of the li rlu and Ih'

the basic intensity of the light varies, with the l~ind of lamp en:-' ploycd. While the range between the brightest to dimmest of them IS great, one a standard exposure has be,en_establishe~ for any giv~~ light sourc " it is usually possible to keep within the rr~axllT1Um an? mll1~mum iutcnsit y of that parri .ular lamp hccaus an inherent latitude m the

of

THE TECHNIQUE

OF PHOTOMICROGRAPHY

FILTER

FACTORS

149

sensitiz.ed. plate~. e can thus have every exposure correct, so far as ~he basic mtenslty IS.co~cerned. Some slight variation can be expected m.all form.s ~f electn~ hght, due to voltage changes on the line. Other mll~or varianons are mtroduc~d in arc lamps by the length of the arc, ~e~lStance carbons of varymg l~n~th, and length of time the lamp IS m. operatlon, but these are neghglble for most purposes. The intenSlty o.f tungsten lamps, on the other hand, varies appreciably with ~hanges m voltag.e, a~d to a lesse~ exten~ with the time a lamp has been lit and the t~tal time It has been m service. Color work is most likely to suffer noticeably when these variations are considerable. ' ~ith respect to the light source, the photomicrographer is much luc~{1er th~n .the outdoor.photograp~1er who must contend with the radical variations present m daylight.

:v

0t

(2)

The Plate Speed

Each type of plate has i~sown speed rating and a basic exposure must b~ determined for each kind used. It is not likely that the plate speed WIll change; hence, when a ratio has been established between several brands, it ~an be .used with a f~ir degree of assurance in determining ~xpos.ure times, WIth t~o exceptl?ns. One is that the failure of the recIprOCIty law n:ay b~ different WIth every type of plate, and the other, that speed ratios WIll not be constant with' all colors of filters. This latter condition, which is the more serious, will be discussed in detail , in connection with the effect of filters on exposure time.

green filter cuts down the light efficiency to a degree equal to t1~eratio of the area of light passed by it, to that passed when no filter ISused. If the two areas were as la: I then the filter would admit only r/IO of the original light energy to the plate. The filter :V0uld .possess a factor of la and whatever exposure would be.reqUlred. WIthout a filter would have to be multiplied by ten ~o se~ure a~ ~qU1valent exposure when the filter is used. otice that m this case It would be the green rays which 'would be. doing all the work of black~ning the plate; but this would make no dIfference, as the plate chosen ISaffec~e.d by gre.en rays as well as by those of other colors. ,T.he same condltlo~1 obtains 'with filters of any other calor charactenstlcs when used WIth plates sensitive to the entire visible spectrum. In each ca~e the filter factor would be represented 'by the ratio of the area of hght passed to the area of the whole square. . But suppose a plate were used which was s~nsit.ive only to blue 11ght. Such a plate sensit.ivity would be grap.hed as m FIgure I ~6C.. At. once ere mse~ted m the it is apparent that if the green filter (FIgure. I 06A) light train, it would cut out the. very port~on of h,ght required to affect the blue sensitive plate, while all the hght which .the filter pa.ssed would be of no use in making an exposure for none of It would regl~ter on the plate. The filter tactor. in s':1chcase would be extremely hIgh, and in some cases approXImate mfimty.. . . It is this condition which makes possible the use of a red lamp in the darkroom 'when developing plates not sensitive to red. On the basis of the above, tWO s~atements can be made as to. the effect of filters on the exposure time: (I) The basic exposure wzt'?out a

:v

(3) Filter Factors


The way i~ which a filter n;odifies the exposure time can be understood from FIgure I06A, which shows the relative amount of light passed by the green filter the curve of which is shown in Figure 88, H no filter '.'!,le.re used the entire area included between 0 and 100 p 'I" cent transn11Ss~onand between 400.and 700 millimicrons might represent th~ total hght ener~y from a g1ven light source, at our command. Assummg that a plate 1Semployed which is sensitive to all rays be twee~ 400 and)oo millimicrons, all available light energy would serve to bU1ld.~p.an image on the p~ate whel~ no .filter is used. The rraph of' the SenSltlVlty of such a plate 1Sshown 111 FIgure lo6B. Inscrt ion of till'

filter must be multiplied by the filter factor of whatever one ~s employed; a~d (2) The factor of any filter will v,ary with the leind of ;plate being used. .. .

To these must be added third condition wl~1ch affects. the filter factor. So far it has been assumed that the quality of tl:e I1ght !~om any source is the same, and eq':1al for all wave lengths 111 the vlsl~le spectrum. This is far ~rom ~~mg true .. Every ~ype of lamp has ItS own particular curve of mtensines f~r va~lOus pOrtlO1~S of the spectrum. Figures r06D and E give an approXlmatlOn of the dIff~rence be.twe.en an arc lamp and tungsten filament lamp. T~1e.former ISstro~1g 111 Vl~let and relatively poor in red; the tungsten I~ Just the OppOSIte .. This heing true it becomes necessary to add a third effect of ~ltcrs in the light train, .i.c., tbe filter actor varies or diIye'rent types of ltgbt sources.

..

SO

'('("';

'l'IWIINIQUI,;

01"

""O'l'OMICHOGRAP"

THE EFFECT OF MAGNIFICATION AND

UMERICAL APERTURE

(4) The Effect of Magnification


By means of the illuminating train and substage condenser, a definite intensity of light per unit area is concentrated on the object. This light is employed to produce the image. When the latter is ten times (in linear dimension) the object size, the superficial areas of the image and object are to each other as the square of ten is to one, i.e., IOO: I. The light, per unit of area, is only 1/ I00 as great in intensity on the image as on the object because it has been spread over an area one hundred times as great. According to the reciprocity law the time must he multiplied by 100 for a ten-power enlargement, over what would be required to photograph the object at natural size. Increasing the magnification to 2OX, i.e., twice the previous amount, decreases the light intensity to 1/400 the original value. This is only one-quarter as much as obtained with a roz magnification. From this we can formulate the ffect of magnification in a general Jaw: The light intensity varies in-

o~----~--__ -=~ __
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Scn8'itivity

of Pamchrornat

t o /'/""

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Light Emittod

It is important to note that this law, as stated, is independent of the objective, eyepiece,' or projection distance, all of which are concerned in the production of the magnification. It makes no difference how the magnification is obtained; the only consideration is the amount of magnification.
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(r) The Effect of Numerical Aperture The numerical apeFture of an objective controls not only the resolution of detail in the image, but the light-gathering power of the lens 'well. The former increases directly with the increase in N.A. 'but he latter increases as the square .of the .A. Assuming other condiions to be constant, the substitution of an objective of double the num .rical aperture Jar one of the smaller value intensifies the light four imcs, This enables the reduction in light intensity attendant on high magnification to be canceled out to a considerable extent, through the I -couipanying increase in N.A. of the higher-power objectives. '1 his effect of increased light intensity with numerical aperture is ubjccr to one condition - that critical light is employed, the aperture If the .ondcnscr (as established by the diaphragm opening) being uhsranrially equal to that of the objective. Obviously, if the aperture of t he condenser is rcdu .cd, the illumination of the object will be cut I vn accordingly. hcohjccrivc .annot gather in light which is not

rod aa in tlu: blufJ. Th


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152

THE TECHNIQUE

OF PHOTOMICROGRAPHY

EFFECT

OF FOGGY LENSES

153

there to be picked up. Should the condenser diaphragm be COil 1 pletely closed, no light would be present. For this reason, the la" 1'(' lating to the effect of numerical aperture on the exposure time must III stated in two parts: The light intensity varies as the square of the N" I,

is only one-quarter of that at f: 4.5: This alteration in the aperture must not be lost sight of when usmg low-power lenses for photomicrography.* .' . . All these factors combined establish the ul~lmate inrensity ~f the light energy upon which depends the production of the laten.t lma~e on the plate. They can be statedrn terins. of a formula whlc.h Will work out to an answer giving the time required to .expose the p~cture. Before this can be done, however, one value. m the equatlO?, a constant K must be known. This constant is to. be determined empirically.' It represents the basic time under a definite setup.

of the objective, with full critical illumination; with less than Filii equivalent aperture of the condenser, the light intensity varies as 1/.1(' square of the effective aperture of the condenser.
This rule applies to low-power regular series of objectives. photographic lenses as well as to till

(6) Effect of Density in the Object


Microscopic objects vary over a wide range in their degree of tran parency or opacity. Naturally this is an important factor in detcrmin ing the exposure time. It is one which is not amenable to a fixed 1:1 \I , other than the general one that the more the light is absorbed by th object, the longer must be the time of exposure. One of the COll'1l11IJIl rules of photography also applies here - that is, aif"'LlJay s expose for Ill/ shadows and let the high lights take care' of themselves. In phot 0 micrography the object (or certain portions of it) constitute the sh.] ows; the high lights are represented by the clear areas surroundin th object. But these two rules give us no mathematical value which cun be inserted into a formula. Relative densities must be detcnnim d largely on the basis of experience. For the purpose of establishinu I standard exposure value, however, little difficulty need be encouni ('Ild in the selection of what might be designated a " typical subject." 'I It might be defined as a well-stained tissue (animal or vegetable) \I hit h in thin section is transparent and colorless in the unstained condit illn Most of these should possess an average density that can s rW;1 .1 standard with which more dense objects may be compared.
I

(8) Effect of Foggy ~enses


Glass surfaces which are allowed to stand ~or some time witho'":t being wiped off u,sllally. devel~p an atmosphenc fog or smol~e. Thl~ is not noticeable m ordinary 11ght but an mte?se beam of 11ght pro .cered on them 'discloses its presence. For this reason all ~on~er:ser lens surfaces should be wiped clean whenever they lose th~lr brilliant polished appearance. !his fog. can affect exposures materially, especially in the blue and violet regions of the spectrum. , '
In calculating the time of exposure from a basic value established on numencal qperture (as is naturally the common practice in photomicrography) when .USIl1~ 10:V lower lenses whose speeds are expressed in "f" values and WhIC~ are eqUlppe WIt Iris dia hra ms capable of being set at lm:-rer apertures, It IS,desirable t~ be able to ex Jresspthe geffective aperture being used, 111terms of ~umencal .aperture. The null'l~rical aperture equivalent of an "f" val.ue for an.y gIven magnIficatIon can be determined by means of 'the formula: M .A.

2f X (M

I) r

(7) Effect of Aperture

Variations in Low-Power

Lenses
I

When a low-power lens, with an aperture, let us say, of f+ 1 used with a long bellows extension its effective aperture appri)\illl;lfl the full aperture (which is realized only with a proje .non distilllll equal to infinity). As the projection distance is shortened, till' 1111111 conjugate focus is lengthened and the effective aperture is tll'l'n"" Id accordingly. It reached its lowest value (in phOtolllit'l'ogl ;ll'h, ) when the magnification rea .hcs I x (full size). The focul lcn rt" is rlu-u doubled and the aperture is j':<). At this :lpcrtlll'e the li 1'\11 illt '1\ It\

This formula automatically takes care of the variable conditions mentioned abov~, and hence offers the easiest rnethod of introducing the ape.l'ture effect 1I1toa ~I~e e~ posure computation. For those doing considerable. w.ork 111 the IOill-powe~ f'~ ~n using various diaphragm openings, a table of N.A. eqUivalents for d erent va ues ut different magnifications can be worked out for ready refe~enc~. "" ' Should one desire to know whether a given setup of magnIficatIon an? f ope~ll1g results in exceeding the theoretical. limit of re.solutIon.' this can be readily ~c~rta~ned by the simple expedient of moving the decimal pomt of the resultant d' h' t1re~ ilnccs to tile right (i.e., multiplying by rooo): This figure sh?uld not excee t ~ mag ~\ification, to meet this requirement, although this does not Imply t~la~ g?od PICt?~~S '1I1lllot 'be taken under the assumed condition, even when the limit IS materia y cecded. li h It should be apparent that the focal length of the lens does not affect the Ig tnth 'ring powcr, rind hen c it makes no chffercn,ec III the c~pos,ur~ whether a lens be 10 111111. (.)1' n lOO 1I11l1., pl'()viliing rh . " value IS rh' snmc In either case.

r"

THE TECHNIQUE

OF PHOTOMICROGRAPHY

DETERlV~INING THE BASIC EXPOSURE

155

B. Determining the Basic Exposure


The basic exposure should be established on a typical slide under conditions of magnification, filter, plate, and lens combination most nearly approximating an average condition for the Class of work one plans to do, If the magnification range contemplated extends all thc way from low powers to very high, an intermediate value would bc around j ooz. The objective required for this combination 'will b either a roz or 20X (40X only with very short bellows). Assuming it ro be the IOX, with the outfit all set for operation, a slide placed 011 the stage, critical light secured, and the image focussed on the ground glass, a test should be made of the effect of various filters on the appearance of the image. One should be selected which seems to giv ' the best effect visually. Let us assume that this is the green filter CB). \ V e are now ready to make a test exposure. When a multiplicator back is available the procedure is as follows: Place the multiplicator back in position, put the slot diaphragm in place, then the loaded plate holder. The number of test exposures which can be made will depend upon the size of the plate and the width of the slot diaphragm used. 'I he 10 mm. slot is ample and provides the largest number of ex posures. If there be something of special interest in the specimen, 11 should be brought into position so that it falls in the area of the slot. I':vc:ry test exposure will then contain the object of special inter .sr. I\s thl' test exposures are made the plateholder is moved along tothe Ill' t position, and so on until all are taken, Test exposure times ;11'(' .1I\\'a\'s made in a geometric ratio; starting with the shortest, each sur (' , ,dIll r l"'1lOs11reis double the preceding one. For the assumed COil dll illll IIf lOO""magnification, roz objective and green filter, wit h il 1111 wuu tungsten lamp, a safe series can be (in seconds): 1,2, 4,H, I , lO, (10. Should the image be quite faint on the ground glass :11111 rh, plate not large enough to accommodate so many, the series 'all hI' , 10, 0, of 0, So seconds, IkVl'loplllel1t of the plate will at once show which onc of rh SIrip had rhc correct exposure. Figure IO? shows a print made frou: Sill h a t cxt plate. The time series in this case was zi, 5, 10, z o, 40, Ho S('(' onds. I':"en the 2 I second exposure made a faint impression 0111111 plall', hUI printing for rhe strip which was correctly exposed (#.1 tit 'Cl Sl'('Olllls) compl .tcly hla .kcns both this and Ih . of second !'It Ill' (TOl'dillgly, ill 111:11 ing the print, it was progrvssiv 'Iy l'''posl'd, 1111 h0111l11l portion rlTeivillg I seconds, the lOp, only I se xmd. Onl

r
, FIG. 107. TEST EXPOSURE TAKEN WITH MULTlPLlCATOR BACK

The object, a diatom, Arachnoidiscus, at a magnification of 500X, taken on HamIIIl'r Slow plate, the exposure being at d', 5, ID, 20,40, and 80 secon~s. , 1'0 show the presence of a faint image in the short exposure stnps, the pnnt was Ilodgcd progressively from one to twelve se~onds, only the bo~tom portion show1111( thc full exposure time, At the ext~eme r;ght a r z-second P7mt of the 2O:second poslIre .strip is shown, m order to gIVe a correct InterpretatIon, of this exposure. )loor picture can he made from, th~ unde~exposed, ID-second stnp by pnntl~g ~or IlIIl' second, wl ile a fairly good prmt IS possible from the 40-second stnp by prmtmg ur uliout two minutes.

rh ' bottom part of the print shows ~he 2O-secor:d strip as it should h , hilt at the top is seen the kind of pIcture resulting fr?m the underposed strips. r roh right is shown a r z-sccond pnnt of the #4

THE TECHNIQUE

OF PHOTOMICROGRAPHY

THE

COMPUTATION

OF EXPOSURES

157

strip. Incidentally, this particular exposure test would not be qllite i representative of that assumed as an average condition. It represents the type of test that might be made by one whose interest lay in the 1 photographing of diatoms. , . . Understanding the principle involved in the making of a test ex-. posure will enable anyone to make such test with an ordinary plate- I holder. Here he does not take a duplication of strips of the same area, but must proportionately expose a portion of the entire field. To do, this, the plateholder slide is withdrawn to allow the entire plate to be exposed, but not completely out of theslot. A computation is made as to the number of exposures desired and the length of the plate subdivided accordingly. If the strips are to be,)et us say, t-inch wide, the first exposure of I second is made on the whole plate. Then the slide is pushed in t-inch and another I -second exposure made. The half inch covered up would have received only one second exposure, all the rest of the plate two seconds. The slide is again. pushed in t an inch and a a-second exposure made. Similarly after each exposuF ' the slide is pushed in and the following exposure is made, doubling the previous time. Marks can be made on the plateholder slide to indicate its position for each exposure. Moving the plateholder slide between exposures may result in some displacement of the camera back and the long exposures may be blurred, .but this should be ignored. Similar test exposures should be made for each type of plate used, unless information covering the relative sensitivity of the plates 10 the type of light used can be secured from the manufacturer. Rcln tive daylight sensitivity is no criterion to follow for photomicro graphic work. , When the standard exposure has been determined, it should be IT corded, with all other data, as follows: .. Standard Exposure - object of normal density Objective - TOX, N.A. 30 Magnification - 300X Filter - B (green), Factor 8 times (manufacturer's data) Plate - Wratten M Light - 500 watt concentrated filament Exposure, 30 seconds (this is the constant, K, required) A data book should be kept for recording all .uch information. will become valuable as time goes by.

C.

The Computation

of Exposures

Having determined the standard exposure time, it becomes a simple matter to compute the equivalent time for any other combination. This is done solely on the basis of substitution of the numerical value of the changed factors. For instance, let us assume the effect of single changes on the time. Having found out empirically that the Wratten Panchromati plate is 3 times faster than the W ratten M, if it be substituted for th latter, the exposure would be
3013 or
10

seconds.

-The orange filter (G) has a factor of 2 as compared with 8 for the green filter. Substitution of the orange filter will mean an e po sure of . 30 X 2/8 or 7t seconds. If we wish to increase the magnification to 400X, using the sail\(' objective, as the light intensity varies inversely with the squar . ()f I h(' magnification, the time required under the new condition would hl'
o

30 X

400
--9

2 (.

300-

l.e., --9000

I6000)

= 53 seconds.
10.\'

. 30

Substitution of a 20X objective with an aperture of .65 for t he N.A. will affect the exposure as has been explained, by
30

30~ (. .09 ) X =r:: i.e., -.65.3925

= 7 secon ds.

Just as it is possible to compute the proper exposure time when onl one variable is present, so it can be done even when everyone of Iht'1I1 is changed. Making all the changes as above at one time the equal ion becomes .

K (base exp.) X filter factor X mag. factor X N.A. factor X plall'


factor
2

. exposure
.302
I

X - 2 X 30 X - X 8 . 300.65 3
--0

2 400

second.

It

Should the object be changed to one more dense, the diff 'I' 'nc'(.' ill the density must be estimated and this factor also inserted ill the equation,

THE

TECHNIQUE

OF PHOTOMICROGRAPHY

SECURING

A SHARP

FOCUS

ON THE

GROU

D GLASS

159

The simplest method of solving the equation is by the use of a slide rule. The relation of the squares can be instantly read off the scale, when the numbers are set in ratio on the C and D scales, thus s:lving the trouble of computing them. Inexpensive slide rules are nvuilublc, they need not be so accurately engraved as when-required for more important determinations. \ Vhcrc certain fixed combinations are employed to a great extent, d:ll:l ruhlcs can be made up for ready reference. These will save reClIllIllIllillg cvery time the same set of conditions is present. 'Ill' question is frequently asked, "How about the use of light IIll'll'I'S?" These have been found so successful in ordinary pho10 rr:lphy and motion-picture work that they would seem ~he answer ill photoruicrography. Various light meters have been put on the market for phoromicrographic work and some few individuals are using Ihem, fior certain restricted classes of work and within certain ranges Ihey cun he successfully used, but they cannot cover the entire range alld :\I't' worse than useless under some conditions. Where the light is int cnsc and fairly short exposures are involved, and where' narrow hunds ill the spectITlln are not being used, they are of value. On the whole, however, when one has mastered the art of computing the t' POSIII'l',he would far rather trust his slide rule than any meter.

it is entirely unnecessary. By the simple expedient of darkening windows and turning off all artificial illumination in the room, focussing and manipulation of the slide can. be carried on without discomfort. When this procedure is made a regular habit it will be found decidedly worth while. (2) Checking Possible Vibration

It may be some time after the photomicrographic apparatus is set 'up and in use before any evidence of vibration can be detected. This may be due to intermittent conditions, or to a failure to detect the type of vibrations which cause trouble. A lookout for this possible trouble should be maintained from the beginning. Occasional fuzziness of pictures, not traceable to any other cause, should be ample-excuse for suspecting vibration. If it seems possible that some is present, one of the best methods of combating it is to place sponge rubber pads 6 or 8 inches square and at least an inch thick under each leg of the table UpON which the apparatus is mounted. On top of the rubber pads place similar-sized wood or metal plates to distribute the load. The table legs rest upon the plates. (3) Securing a Sharp Focus on the Ground Glass

I)

I//lc'1'1I11I1C()/IS

Items Related

to Exposure

Ill' I\1111Il'I'OUS little details related to some part of the photoI Iphl PIIICl'SS which do not amount to much in themselves, but III I h .1 'It' ':lIt' will be found to have considerable bearing on Ic It I 11 \ III IIll' work. It is necessary to be constantly on the I Id '11 und means for improving one's photomicrographic tlC,lllU1hlll 1111011 h Ihe upplication of some of the things which exI I III I -vcu I.
I

tb I

f)f)'1II

during Exposure

point cd out that means should be available for where Ihe work is bcing done. There are two I I1 I )11 ' is I he possible interferon .c of extraneous light 1111111 1111111lit !Ill' ohjt'l'l, 'specially at low IIl:lgnifi .nrion. The I I 1111\ 'lIil'lll'l' of r()cIIsill r propcrl: in n hrighlly lightt'd I h 11\ '11 'lit :t Incussiu r clot h over I he head is possible,
'11

It ,h h'
Ih

100111

It is extremely difficult for the unaided eye to determine when the image is in perfect focus. For this reason focussing glasses are provided by the manufacturers. The common form is a o-power magnifier mounted in a tube support which can-be rested aga.inst the glass. The magnifier must be adjustable to adapt it to the eyes of different individuals. The camera is either provided with a clear glass focussing screen which can be substituted for the ground glass, or a small cover glass is cemented to the center of the ground glass with Canada balsam. This eliminates the ground surface and the glass, appears as though clear at this one spot. The eye cannot discern an image on clear glass, but a magnifier picks up the image just as the microscope eyepiece does that of the objective, even though no screen be present. The clear glass focussing screen, in combination 'with the magnifier, provides a critical focus, especially with low and medium powers.' Ir is subject to one limitation, however, which has made it responsible for I)()or pictures in many cases, because of lack of information regaJ'( ing it. It is very important that the utagnifier be to cussed on tbe

160

THE TECHNIQUEOF PHOTOMICROGRAPHY

CHaos I For long not matter, where eyethis respect, a mark on ground or

THE PROPERFILTER

r6r

glass surface corresponding


but for short projection pieces arc not used, this Ihe magnifier must be I hl' "round glass; then clcnr glass.

to the position of tbe plate.

h .llows lengths and for medium

and high powers it does distances and low-power lenses is imperative. To avoid trouble in adjusted by focussing sharply on it is ready for use with either the

under a given condition. 0 one need be ashamed of his ignorance in this respect, for this is a problem likely to st~lmp th~ best of ~s. .It is true that experience in the taking of many pIctures IS a material aid in the majority of cases, but it sometimes happens that ~he color ~che~e of an object does not seem to be amenable to any possible combination of filters. .

\ Vhcn the magnification is high for the aperture being used (i.e., Ill':1r t h ' point of empty magnification) the focussing glass will work bl't llT Oil t he ground glass, because a six times enlargement of an image alread), on the" empty" side is so fuzzy, it is of no use for deter~lIillillK the .orrecr focus .. Under this condition a far ~etter met~od IS to discard the 6x magmfier altogether and use an ordinary readmg r/ass of I \VI) or three power, on the ground glass. Focussing becomes quite tasy under this method. (-/-) The Use of Diaphragm Masks in Front of tbe Plate
J\s a rll.le, the larger Out.fi~s are equipped with metal diaphragm IIIasks which can be placed m the back of the camera, directly in frollt of the plateholder. The masks are made with different-sized ('i Iell'S, ada prablc to various sizes of plates. finished as a circle to si mu:1ppcar?nce, the mask. is valuabl 7 in circumscribing the I \ I1I Ihl' form desired for the pnnt. In this way one can be sure of \ hut i ill~'lu~lcd. But ~t often happens ~hat a reetaugular or ~quare 1 I 1 ul: '1'101' III.that a I:l~her magmfic~tlOn or a gr~ater area IS proI d IIdl:r IhIS condition the mask .IS bette.r omitted. Examples 11 lu I'd III the plates of Chapter 8 illustratinp the use of the full 11 of Ill', platl". Thcn again,. the.re a~e occasions when only a ulm 1'11111 I required and the object ISa dispersion of small particles. 111111111 ,Ihl' circular, mask, if the entire plate is covered by the II I huicc of area IS offered, from which to make a selection 1111111111 the mnt crjn] in the best possible manner,
111 ' till'

I' 01' \\'o.rl- where the print is preferably


visual

FIG. 108.

LONGTTUDINALSECTIO " RHIZOME OF FERN

Three views taken with multiplicator back, showing the results obtai?ed with three different filters. Left - A filter (red); Middle - B filter (green); RIght - H filter (blue) .

/100\;"
I
III

tht' 1 roj1t'r Filter


1

hili 'ill ch:lr:H'llTislics


11 r d u to how

of flit .rs :lnd their <:fTect on the .x have h ' '11 covered already, hut t he novice III:I\' he IIIOI'L' to ilia I e a filial decision Oil which to use

of a couple of simple exa~ples will help to .explai~ the si~Figure r08 shows three vle~s of a fer?- ~hlZome 111 10I~glrudinal section. These were taken WIth the multiplicator back, usmg red, grecn, and blue filters respectively.. The section w~s stained with fuchsin and methylene blue, the scalanf~rl11 vessels takmg a .deep red color, the rest of rh lis arc pr dominantly blue but WIth some residual red [roru the fuchsin and SOI1IC grcenish areas, caused by re-

Study uation.

-1
THE TECHNIQUE aCI OF PHOTOMICROGRAPHY PICTORIAL COMPOSITION IN PHOTOMICROGRAPHY

ions between the methylene blue and the cells (the staining being dune on fresh-cut sections without previous fixing), .'r~l ' red filter (i,e" used on the picture on the left) has completely -liruinutcd the scalariform vessels but shows the other cells. The I" '11 (ilrcr (center) accentuates the red vessels and also brings out

Though the insect has been cleared and mounted in balsam, its color is still a rich brown, The red filter reveals the detail structure beautifully; both the green and blue are hopeless, But the red filter is deficient in one respect, What has become of the beal: appendages evident in the other views? In the green filter picture they show with the proper amount of contrast, but all the other parts are too dark. The trouble lies in the paleness of the color in the appendages as compared with the depth of color in the rest of the body, ' The 'appendages are -practically invisible in the red light, Expressed in general terms, it is always a problem when a filter of a certain color must be employed to show detail in one portion of the object, and yet. this same filter either renders some other portion opaque or completely transparent and invisible, Sometimes it requires two pictures to tell the whole story, When this is impossible, a jugglin.g of the exposure time, over- or underexposing, as the case may require, supplemented by over- or underdevelopment, may effect a compromise, ' (6)" Pictorial Composition in Photomicrography

FII"

lot),

[f1~A)) AN)) BI1.AK 0[0'

Corrox

BOLL WEEV[1.

I \

[111((111 with

f hi,

I ,eft

mulriplicator 1\ filter (red);

back, showing the results obtained with three Middle - B filter (green); Right - 11 filter

nicely. The blue filter (right) also shows the vessels !l'ss contrast than the green, There is enough grecn II! 01 her cells 10 ilia kc them show nicely but with lessen 'd 'Ill! Ihis specimen the green filter is obviously the best, I I!' calal'ifOl'lll Vl'SS 'Is stand out better. This does 1101 11011 PI'Ohlt'Il1 as 10 which filter to USt, 111I1 ill Ii III'l' I ()(), II shows I hc hl'ad :lIld hcul of I Ill' cot J.Uj' '~11 11 0 pholo ruph -d wit h : h . rvd, 'Tl'CIl, :111\1hlue iilrcrs.
ill!

It may sound a little incongruous to refer to' pictorial composition in photomicrography, but there are times when the composition of the subject within the picture area is a matte}" of importance, No hard and fast rules can be laid down to serve as a guide but attention to this aspect of the photomicrographic problem will usually produce, higher quality pictures, Among the type of. questions that should be considered are: . r (~) How much should be included in the picture? (2) Where an entire object is to be shown, how much border is required around it? (3) Which way shall it be turned, horizontally, vertically, or diagonally? , (4) In case' of a poor area which must be included in the picture (an artifact, torn section, dirt, out of focus portion, etc.), where shall it be located in the picture space, to be least noticeable? (5) Where only a segment of a circular object can be included, should the outer portion be located at the top, bottom, or side? (6) Where two or three objects are to be included in the field, how should they be placed to convey the impression of balance in the

pi .turc?

THE TECH T1QUE OF PHOTOMICROGRAPHY

OVERCOMING CURVATURE OF THE FIELD

165

'tany sllcl~ q~e~tioI~s n:i~ht ~e cited. ~n artistic temperament is a rrcnt help In glVmg individuality to one s work. Two adjuncts of IIll' la r re research microscope aid in obtaining the best answers to these 'IlIt'liIlIlS. Onc is. the rotatingstage, by means of which t~1eobject 1111 ht' I11 r~ll'd to (hffe~ent posltlons to study the effect. The other is I fill' 1I11I111I1l me .hanical stage. These, combined with an adjustable h 1111 \ , und a little patience in examining the image on the ground 1I , \ III 11lIally result in a better picture.

S07ne C07n111,On and Unusual Problems in Photomicrography In addition to the common; everyday problems confronting the microscopist in his photographic work, others more or less unusual in nature may arise from time to time. A brief consideration of some of these, and suggestions as to how they can be met, may prove of value. One of the most bothersome is likely to be curvature of the field, which is al:yays present with ordinary eyepieces. When the center of the field is in focus, the outer margins are all blurred; when the focus is altered to make the outer zones sharp, the center is no longer in focus. Pictures taken under either condition are not satisfying, (I) Overcoming Curvature of the Field

phllrc 11 11 il'l'lIgrapher who desires excellence, one of the most I pplllllllll ~'"lldil ions is th.e presence of some defect at the very pi I ' ~ III Ilie hd~' where the pIcture must be taken. Shrinkage of the II 11' III It ,IIIC,II~ IS IISII.:' 11 Y not very ?bj~ctionable, provided no tearing 1~lls re lilt xl. .' he artifacts most objectionable are pieces of dirt, knife lin 'S, 1111'1I sccnons, folded over or buckled sections, and the like. For ~'isllal \~'ork these arc not objectionable; the eye is capable of discountII~ t hell: pr~sence and concentrating on the point of interest. But a prcturc IS diflcrcnt: by some process of psychology the very thing rhur shouldn't ~)eseen becomes the center of interest, or, at any rate, d 'I ra~'Is arrcnnon fro~l the rest of the picture. Elimination of artif.I'~ ISIlot ahv:lys P?ssIble, but numerous expedients may be tried with I \ I '\~' 10 rcrnng rid of them. Among these are a slight change in IIII nili ':~tlOIl to throw them out of the field; decentering the main 1"1111 IIf 1111 'rest; use of a.square or rectangular mask instead of a cir11111 1I1l'~or at least placing them in some position in the field where I "I 'a'l likely to attract attention. IthCIII h I' -rouching, as ~ommonlr used with ordinary photographs, I I I IIlIh,le '" p.horOllllcrographIc work, yet if it is solely for the I I of '11I1~1I.latlllg, or rendermg less conspicuous, undesirable arI 11 I ,"s"f~ah.le. If ()~leunderstands retouching, it is usually an 111111 I ,Ill '111I."lat(.' knife scrat .hcs, tears in tissues, by the use of I III 11111', I)11'1 spots can often he eradicated by careful use of n I III 'h '1I1l he rradll:t1lr built lip ~lI1rilits intensity matches rh ' "'I~k'rrulll I, 111111' as Ihl' prlllllllg dTecr IS cone 'riled. In the instance I Ih IlIrI,lIl1 SIIITIIIIIHlillgan isolated objccr, hlo('l<ing OUI of , I I I 101llld nmv hl' Ihe cnsi 'SI solut ion. \ Vhcrc the buck I I PilI' white, this is no: ohjccrionuhle.

I Ih

One worker who toole up photomicrography told me of his discovery that everything could not be sharply in focus at the same time. He had laid it to the achromatic objectives he was using and purchased a set of apochrornats and compensating eyepieces. His next discovery was that the trouble, instead of being corrected, was worse. It was then that he sought my advice. He was right in his second discovery; the trouble is usually worse with apochromats than with the less expensive lenses., Conditions are not improved by the use of projection eyepieces, for these also have a curved field. There are, however, several methods by which fairly flat fields can be secured. First, one may employ a long bellows extension with a 10\V-or medium-power eyepiece. The entire field is then quite large in diameter, and only the central portion of it is used. If one lacks a long bellows, a high-power eyepiece will accomplish nearly the same result. Combining both is better yet. These methods introduce some , complications, in that the magnification is increased at the same time. To lower the magnification. will probably require the use of a lowerpower objective with a lower numerical aperture. This in turn lowers the resolution and may even bring one into the realm of empty magnification. Sometimes a compromise can be made by a partial correction t hrtlugL these means, supplemented by a focus on a point midway bCIwccn the cenrer and circumference of the field. J\tosr manufacturers now make a series of so-called flat-field oculars for visual work which arc superior to the regular series of Huygenian eyepieces. 'I hest' can he used 10 some advantage for' securing flatter

THE

TECHNIQUE

OF PHOTOMICROGRAPHY

OPTICAL SECTIONING

fields in photomicrography, but they arc still not perfect. By far the best solution lies in the use of eyepieces such as the Homals, which have been developed especially for photographic purposes." The Iicld produced by these is beautifully flat. Figures 110 and I I I illus-

high cost of the set of eyepieces required to cover the complete range of objectives, , (2)

Optical Sectioning

O~1~ .of the most ~aluabl~ assets 'Of rhotomicrography lies in the possibility of portraymg a smgle plane ~n a transparent object of ap-

FIG. ) 10.

GI..A

J)

Cer.r.s

IN TNT" TINE 01,'

E(;T It S

Tnk

'11

with a compensating ocular, and showing the curved field .hn ract 'risli(' nil ordinary cycpicc s. Only the .cntrnl portion of the f .ld is in focus,

of
FIG, III

rrurc the diff r n .c in rh app arancc of the same Iicld when tah'n with an ordinary COI1lP .nsnrin 0 ular and with a I I 0111 a I. Thl' only fly in t h oiru mcnt for SOIIl " in this solution of t h ' prohl 'Ill, lit's ill t h '
111111111"

SIII1I\'slIl>j' 'I' as rhc P'' vious figure, but taken with n photographic whi .h gives n flat field over the 'mire area.

cy 'pi 'C' (1Iol1H1I).

Ilol1l:ll~ Ill'" uSI'I"" fOl' l'islIlIlllIlI'lHISI'S. ~illl'l' t lu- ')'l'poilll II!IOIl' it, II~ ill thl' 1'1I~" of nrrlinur '" 'l'piv '('S,

li,'~ ~Vill,lill t lie I') l'pil't"',

pl'lTiahl' rhic] ncss, without serious int 'rf 'I" '11" [rom lny 'rs :1I)()v' :lnd below. This is lmm n us optical s('('ti()llill r. The only requisite

168

THE TECHNIQUE

OF PHOTOMICROGRAPHY

OVERCOMING CONDITIONS IN THE OBJECT

for its accomplishment is the employment of an objective of high numerical aperture. It is not always necessary to use the very highest possible aperture; it must be adapted to the extent of depth permissible with a given object. In every case the aperture of the substage. condenser must also be \1igh, preferably corresponding to that of the objective, and the light must be critical. If desired, a series of optical sections can be made showing progressive changes in structure. For further visualization of the structure, it is possible to make prints of cuch picture in a group of serial sections, on lantern slides, as transpurcncics. These, when piled together in proper sequence, provide a h .auriful three-dimensional effect. For the depth to correspond uccurarcly with the magnification the distance between the progressive st cps i11 the series should be computed so that it is related to the thickIH.!SS of the glass of the transparencies in the same ratio as the surface cnl.u gelllent, For instance, if a magnification of IOOOX is employed, change in depth of each view should be 1/ I 000 of the thickness of the transparency glass. In measuring the movement on the graduated fine adjustment of the microscope, to secure this actual depth, the refractive index of the medium in which the specimen is mounted must be taken into account. This is usually around 1.5, hence if the desired thickness be
I.2

(4) Ooercoming Conditions in the Object


One cannot photograph many slides without discove~ing the existence of numerous conditions which are detrimental to Ideal results. Sections are found which are wavy and cannot be got in focus everywhere at once. Some sections are cut very thick; others are stained too darkly, or hardly at all. The glass slips used for mounting are often w9-vy and uneven to the extent th~t specimens such as .b~ood smears, bacteria, etc., are not everywhere m focus at the same tln:e at the very point where. a particular field is wanted. Some unstained objects possess a refractive :index almost identical wit~ that of the media in which they are mounted. These and others of like nature are continually cropping up and each may require the empl?YJ?ent ?f new techniques" or modifications of ol? In gener~l, familiarity WIth the fundamentals already discussed WIll suggest SUItable methods to meet such situations as they arise. . Wavy sections in histological and pathological work are extremely common, and sometimes tax the ingenuity of the photomicrographer to the limit. The novice may recognize that something is wrong, without being able to detect the cause of the trouble. When one part of the field is in focus, another is out. The focus can be changed to bring the latter in sharply; then the .former is fu:zy. Several methods of eliminating the trouble are poss1?le, dependms- upon the amount of leeway one has, in the magnification, area required, etc.: Suc.h ~\cvices as raising the magnification to red?ce the area?f the ~eld; shifting the area' slightly; and occasionally US111g an ~yeplece WIth a. c?~~ed field which compensates for the curved section, are all posslbllrt~es. sing a lower-aperture objective, as describ~d abov~, for ~ecunng depth of focus, is one of the best methods. ,ThIS necessltate~ hlgl: eyepiecing or increased projection distance to restore the m~gl1l.ficatlon to the desired amount. Uneven slides cause trouble only 111 hIgh-power work with bacteria, blood, and similar surface smears. The effect is identical with that produced by wavy sections and the method~ of .ovcrcorning it are the same .. Sometimes ~wo or more cel~s ar.e d~slred to he in focus at the same time, yet a hIgh-aperture obJectlve. IS re-, quircd, In this case, one or .the ot!ler ?f the cells must be sacnfic.ed. Thick sections can be handled 111 either of two ways, depending IIpon rh result desir d. One is to use.a lo.w-aperrur? lens with ~~1I~sidcrnh I ' depth of Focus to get ev ryrhing 1I~ the section sharp. I hiS is usun 11y t he h tt l' I'IlICthod for low- and rued IIIIlI power work. \ Vhcn

microns, the registered movement must be ~

r.5

or .8 -

micron. * The transparency arc to be superimposed.

prints must be rather light if many levels .

(1) Securing Depth of Focus This is exactly the opposite of optical sectioning. The rnann I' of its accomplishment therefore lies in doing just the opposite of what OIlCwould do for optical sectioning - that is, use lenses of the lowest possible numerical aperture. Magnification must be secured hy high power eyepieces and long bellows extensions. Reduction of the suh stage condenser aperture can also be resorted to, up to the limit where diffraction effects begin to intrude. Though many conditions in transmitted light work call for Ill(' greatest possible depth of focus, this requirement is st ill mor . (.;OIlIIIlOIl for opaque objects with very uneven surfaces.
Only slands wit h rhc v("ry (inesl of (in' ndjusuncru
sIIIIIIIIIIOH'III(III.

('illl

hl' l'\Pl'l'll'd

10 111('('1Ihi

THE TECI-I IQUE OF PHOTOMICROGRAPHY

INHERENT

LIMITING

CO DITIONS IN PI-IOTOMICROGRAPHY

high power is permissible, or required, the optical section method will solve the problem but care must be taken to focus on the most important plane in the section. Dark objects and sections that are intensely stained rnust be given

fraction is objectionable, as a general proposition, we must allow it here, or not obtain a picture at all. Figure 112 illustrates the appearance of a diatom (Auliscus) at a magnification of rooox, under three conditions of illumination. The three views were taken with a multiplicator back and the only differ.ence in the conditions was in the aperture of the illuminating cone. The first view (at left) shows the result obtained with a full cone, corresponding to the aperture of the objective. In the second view the aperture was closed to one-half that of the objective, and in the third the aperture was one-half of the second. In order to compensate for the change in light intensity, the second picture was given an exposure of four times the first and the third was increased to sixteen times the first. The progressive increase in the contrast is marked, although one condition has not been changed - the structureless parts of the diatom are not darkened. They still possess the same- tone as the background. It should be pointed out that whereas the stopping down of the condenser affected the exposure time in the ratios of I: 4: 16, the resolution does not suffer to this extent. The resolution is reduced directly as the effective aperture of the system is changed. With a full cone of illumination in use).
I

the aperture is

+
2

I,

or unity (i.e., that of the objective

In the second case it is

+t =
,2

.75, while in the third it is

. -+ .. -i = . 625,
2 DELl EATIO OF DIATOM STRUCTURE. TURE OF ILLUMl ACCOMPLISHED THROUGH REDUCED AJ'I':lt-

(5) Inherent Limiting Conditions in Pbotomicrography


In commercial photomicrographic work, one is frequently asked to meet impossible conditions in producing a picture. For instance, three requirements will be set: a magnification of a definite size, a definite field to be included, and the picture to be made on a 3i" x 4" lantern slide, for lecture purposes: Such requirements are subject to the operation of a law analogous to the phase rule. Any two conditions can lie ~xed, but the third must be variable. Suppose the stipulated requirement to be that an object r mm. in diameter be photographed at IOOOX on a lantern slide plate. One millirneter multiplied by 1000 is IO()O111111. or about 40 inches. Therefore, if the object and magnification <Ire definitely fixed, the final picture will be 3! feet in diameter,

ATION CONE

Left - Full aperture, equal to that of objective. Middle - Aperture reduced one-half. Right - Aperture reduced to one-quarter of objective.

ample exposure, supplemented, where possible, by the use of a filt 'I" which will reduce contrasts to a satisfactory degree. nsraincd, colorless objects mounted in media having refract ivc indices close to that of the objects, require a reduction in thc apcrtuIT of the cone of illumination to produce the necessary contrast. Such ohjl'cts arc said to posscss 1000v relief, or low refractive ind .x di(f('1' cnrinrion. Critical illumination with a full .onc from Ihe condcuscr must he modified in order to int reduce di!Tfaction, lrhou rh dif

I72

THE ,TECHNIQUE

OF PHOTOMICROGRAPHY

Till': liSP:Oil

I'

1111I11\ IllCoIl lA(; Ill'ICA'1'I0NS

which obviously cannot go on a x 4// plate. Either the magnifica tion or the part of the object included in the picture must be rcdu 'cd to about I/I2 .the stipulated dimension if the most important require ment is that the picture must be shown as a lantern slide. It is SOIlI' times hard to make a non-scientifically minded client see this point. Another limitation, not often appreciated by microscopists them selves, lies in the impossibility of photographing, in one view, a lar re object of considerable depth and at the same time revealing minute structure which requires a high-aperture lens to resolve. ITere till' limiting conditions are diametrically opposed to each other. On cannot have great depth of focus, a low magnification, and great rcso lution at one and the same time. The answer is usually easy, ho ever - take two pictures. One picture can show the general view, the other the specific detail characteristic of the object.
(6)

3r

both horizontally ;1I1dvert \(,,111\, It t t"luin:s only mechanical skill to assemble the pictures. ., his c.m ht, done b means of adhcsizc strips on the. back, the who I ' hcin r mounted on a large card as the filial operatlon.

(7) The Use of Extremely

lIigb Magnifications

Sectional Map Pictures

It was suggested or inferred in the previous section that it is im possible to give a client what he may require in the way of lilagni(ic:\ tion for a given object size, provided no limit is placed upon the sil'.e of the picture. Naturally the response to this is that the camera limits the plate size, and that therefore a forty-inch-diameter picture is jll~t as impossible as the other requirement. This is true, if wc arc to ht, limited to one exposure. But just as the aerial photographer can 111:111 a whole country by a series of overlapping views which C<1n suhsc . quently be matched and combined into one composite whole, so till' photomicrographer can produce map pictures of any size by SIlCt'CS sive exposures which can be matched together the same way. The author has made numerous pictures of this type, extending to scv '1':11 feet in diameter and composed in some cases of upward of H hundred separate exposures. To do this requires an accurately graduatcd nu' chanical stage. A careful layout of the entire object must be 111:ldl'; the size of the included square of the field at the dctcrrnin xl 111:\ 'nifi cation computed; and the settings on the verniers of the SI:I"C re corded for every square. The light and all other conditions must be constant, and the t'\ posures and developing conditions on every plate he idcnt iea I. S()llll' overlap should be allowed on the squares to take cm,' of pos~ihk ill accuracy in the setting of the verniers; oth .rwisc one Illi rill find .1 thin strip of area missing at some pia' '. "he pi 'llIrt'S 11111S1 111:11('\1,

There 'is a fascination, for some individuals, at least, in the produ 'tion of pictures in the realm of super-power magnifications. It prohably results from a desire to see and reveal structures that have be n beyond the ken of others. Usually nothing results from such pie tures and they are only mediocre in quality; nevertheles , there is :I distinct value in encouraging this class of work, for in the end we 111:1 derive something out of it which will further the cause of science. Some notable work has been done along this line by several workers. The limiting factors in high-power work are two: the basic opt ic:!1 laws and the perfection of the mechanical and optical parts of I ht' microscope and equipment. ' We have seen (Chapter I) that there is a limit in effective mag'lIifl cation, beyond which further increase results in "empty magllifi('a tion." This limit is established at roughly 1000 times the numcrirnl aperture of the objective, based upon a circle of confusion of z so microns. Some have even insisted that 250 microns is too large a valu ; thal the ultimate limit should be set as low as 75 microns. This low figul'c, based upon the existence of a few hypothetical individuals posscssin r , abnormally good eyesight, is logically absurd, in the light of actual practice, for it is equivalent to saying that a 1.4 .A. apochrornat, our highest available aperture, cannot be expected to perform, at the strictest standard of useful definition, beyond a 'maximum 'lI1ag'lli/ictl

ti011of

420X!

It is not tha-t there is anything wrong with the theory; the arbirrnrv standard of excellence is merely set too high. The same criticism ap plies, although to a far smaller degree, to the 250 micron limit for the circle of confusion, 'which establishes a maximum useful magnification for a 1.4 . . aplanat, at 1400 diameters. The t rouble with the entire resolution formula, so far as it applies 10 the maximum useful magnification, is that it does not tal c into account t h ' limitations inherent in the human eye, outside the area oJ
111'1/ I c

visitrn.

174

THE TECH

IQUE OF PHOTOMICROGRAPHY

THE USE OF EXTREMELY

HIGH MAGNIFICATIONS

175

When we say that the eye can resolve two lines at a certain minimum spacing, when they are located ten inches from the eye, we mean that they can be resolved at the point of best vision, the fovea. Away from this area, vision is relatively poor and lines many .times more widely separated cannot be resolved. This is the reason the eye does I10t relish being worked continually at the-limit of vision but prefers an ample margin over it. For instance, if one examines two editions of the same book, alike except that larger type is used in 'one than in the other, he does not say of the latter, " I do not like tfiis edition because I cannot read the fine print," but he says of the other, " I prefer this one because of the larger type." Just so with a photomicrograph: all the detail may be present in one at 1400X which is procurable, but it may be so fine that much of the finer structure is unnoticed. Enlarge the same picture by two, and it becomes a pleasure to look at it. To illustrate, Plate 11 (page 313) shows, a section.through the cochlea of a guinea pig. It was taken with a Planar, stopped down to f: 6.3. The magnification is 2 5x, slightly more than would be permissible with a 75-micron limit on the circle of confusion. To be sure, the picture is beautifully sharp, but can it, at this magnification, tell us all about the structure shown nobicb the lens used is capable of revealing, or as a matter of fact, actually has put into the negative for us to see? The convolutions of the cochlea have a bridgelike membrane through them supporting the organ of Corti, the nerve element which picks up sound vibrations and converts them into energy for transmission to the brain. There is also a second delicate membrane (the membrane of Reissner) extending across the convolutions. These can be seen in the micrograph, but the best human eye known to science cannot learn much concerning the structural details of the membranes, from a superficial examination of Plate 1 I. The best way to answer the question raised is to make an enlargement directly from the negative of Plate 1 I. Figure I 13 shows such an enlargement, seven times actual size, resulting in a total magnification of 175x! T11is enlargement is so great that the grain of the plate is evident and detracts from what the lens itself would produce at the same magnification. Obviously there is considerable empty magnification present, but the net result is a greatly enhanced apprcciation of the structure. The individual cells arc dis .crniblc with as '. Although this illustration is based on a low-power lens, Ihe same

principle applies all along the line to the very hig?est-power obj~ctive. It is apparent that there are two ways of lookmg at the question of the maximum useful magnification provided by any given lens.

FIG, I I 3. SEYE TIMES ENLARGEME 'r" FROM EGATIVE 01' PLATE 1 I Showing a marked increase in the strllctnr~l ~etails revealed, rcgar?lcss of rh ',r.oll sidcrablc CI11pty magnification and plate gram introduced. The ultimate l1laglll(I(':\ tion is 175 diameters, .

\t

There is the theoretical limit, which can be stated in math cm <11 icnl .rms, and the practical limit, which cares nothing about theory bUI is interested only in results. \Ve must therefore disregard all all 'l1IplS 10 )lacl' a limit on ult imat 'lll~l milicntion on t hc husis of theory, and cstnh 1ish :111- indl']ll'IHkllt st.mdard.

THE

TECHNIQUE

OF PHOTOMICROGRAPHY

LOW-POWER

PHOTOMICROGRAPHY

BY TRANSMITTED

LIGHT

177

For practical working purposes, it is a safe law to follow, that the effective limit of magnification with any given lens is that where the image ceases to reveal additional detail in a manner easily discernible to tbe normal eye, at the distance of best vision. , . Such a limit extends the working range of objectives considerably. St~ldy: of many o! the ,plates in Chapter 8 will show the practical apphc~tlon of m~gmficatlons exceeding the theoretical limit of resolution. 10 take high-power micrographs requires, first of all the best quality of objectives; but moder,n ~~D:ses, as.tu~ned out by aU'reputable ?lanufacturers, have more possibilities built into them than are utilized by ave~age microscopists, Hence the limitations imposed on the photomicrographer are often set by conditions other than the objective. . It is necessary, for utiliz~tior: of th~ utmo~t i~ resolving power, to ?11the condenser when usmg immersion objectives having apertures 111excess of 1.0 .A. Amor:~ the factors influencing results are: freedom from vibration, the stability ?f .the ~ne-focus.sing device, the duration of the exposure, and, the vananon 111focus ~n~rod~lced by a. change in temperature ?urmg the exposure. A vanau?n ill the relationship between the obJect and the lens, of one-half micron (1150,000 of an inch) durinz an exposure, is sufficient to spoil a picture completely, when a highaperture lens is being used. Failure to obtain the results desired, especially when the picture does not appear to measure up to the quality of the image as seen on the ground glass, can usually be traced to one or the other of these factors. (8) Superimposing Eyepiece Scales and Rulings

a superimposed scale, the first thing to be done? after ~stablishing the projection distance, is to focus the scale until the lines are sharp, before focussing the image. " " (9) Photographing Objects Mounted in Fluid

Unusual conditions are often present when objects mounted in or suspended in fluids must be photographed. A microscope in thc vertical position is essential. This generally implies a vertical camera. Sometimes, with minute particles, Brownian movement may. be present. This requires a fractional-second exposure to stop motion. more frequent trouble arises when an immersion lens is being lIs~d, because the viscosity of the immersion oil exceeds that of the fluid. Every attempt to focus on a fine object results in a movement of th ' object, because of varying pressure on the cover .glass. Und.er t,hest' conditions it is usually preferable to employ high dry object IVl'S, which should be of the type that are adjustable for cover rlass thickness. Low-Power Pbotomicrograpby by" Transmitted Light

It is frequently desirable t~ superimpose a net ruling, pointer, 'Or e.ngra:red scale o~ the photomicrograph for showing dimensions, particle SlZe,.or deslgna~~d a~eas. ~he ?n~ place in the optical system ~here this superposltlon IS poss:ble ,IS111 .the plane of the eyepiece dlaphrag~l1. .A micrometer eyepiece IS ~eslgned to accommodate any s~ale wh.lch IS to be. seen at the saI"?e time as the ~bject. It is pro~lded ~lth a foc~ss111g eyelens which serves to bring the scale and 1111age111focu~ .slmultaneously. In photography it is important to note one condition - when the scale is in focus for visual worl it is' not in focus for the picture. Moreover, the focus of rhc s '" I ' V:l ril's with every change in projection distance. Therefore, where usin r

For convenience, the line of demarcation between medium alld low-power photomicrography c~n be set at the point wl:cre. sill rlc phot?graphic lenses must be substituted for a low-power objc .nvc alld eyeplCce. . Though the fundamental principles applicable to photomicrograph in general are not different in the low-power field, there 'HC a few spccial problems introduced by the changeover that warrant COli sidcration. In the first place, it is at this point tha~ the Kohler method of illumi nation for low-power work should be introduced, as constant stand nrds for exposure can then be set up and maintained throughout IIll' entire series of microphotographic lenses. To apply this method of illumination, each lens I11l1S.t have its own condenser. This condenser has the same, or substantially the s.uu, focal Icngth. as th~ photo&raphic len~, for it is .1()~atcd dir 'ClI Ill' ncurh the object slid, and Its focal pomt should he 111 th~ plan: o~ ~hl' 'diaphragm of rh lens. The diaructcr of the c(~n~lcnser ISthe 1111111111' di:llllt'Il'r of the field that can be cov 'I"xl, provl(ling the hole t hrou rh thl' IllinoSl'Opeslage is :1I11pk which is nOI alw:lYs the (,:I.se\\'I~tll ~11l' lun 'eSl focus lenses are hc:illg used. I'iillkr low pOWl'l' illuminnrion

THE TECH

IQUE OF PHOTOMICROGRA_PHY

PHOTOMICROGRAPHY

WITH

INCIDENT

LIGHT

I79

requires the use of a diaphragm located between the lamp condenser an.d t~e substage condenser.. The lamp condense- is focussed upon this diaphragm. so that the rays cross at tl11Spomt. This diaphragm is the .apern~re diaphragm and It .should alw~ys be opeJ1 sufficiently to ()bvI~te diffraction effects, while for maximum resolution it should prc.)~~dea fu?l cone, equal to .the N.A. of the photographic lens, to the (:I~Jec~. This can be determined by lookin.g at the rear of the photogl aphlC lens, ~xact1y a~ with. regular objectives. ,~ alllens~s m the entire series have the same aperture ratio), when :I suu a.ble .dlaphragm aperture has been established for one lens, it can h~',1I1:II.nr;lmed for all, and changing lenses to provide the desired mag~l.JI~('at~ol~ does not affect the ~xposure time, ~n so ~ar as the aperture I~ ~()J~c:el ned. The exposur~ tlm~, however, IS subject to the law reLitIIlg ~o.the effect of rnagnificarion, Just as with high-power work. ,." ;~(,I~I~r,lon, as has already. b~en pointed .Out (page 152), exposure ~IIl1es:11 e ~(f~cted by tl~e v~natl?nln effectzve aperture, resulting from ,lilY change 10 the projecnon distance. ~1.th()lIgh it is possible to use the Kohler setup without the interP~)sltlon of a parallelizing auxiliary condenser between the aperture ~iI:lpllr;l~'I~land substa~e co~de?ser, it is advantageous to employ it. I he C(hc,.ency of. the IllumInatIOn system is raised by its use, as it is t ht',~lpossible to direct a~l ~h~ rays i~to the substage condenser. I or low-power work It IS ImperatIve to employ a shutter with ac('11 rat cly controlled ,fractional-second speeds, as very short cxposu res al'l'. the rule, especIally. when high-intensity lamps arc used. It is l!t's,.r,al,)1c to ,make a. series of ~est exposures on a low-power basis, .111111.11 to that descnbed for high-power work. In this connection It IllIlSt be.remembered that any change in the size of opening in the ,Ip -nure dl.aphragm afec.ts the exposure inversely as the square of rhe lip -rtu re diamcrcr, For Instance, if the established standard opcnin r htls h 't'll se.t at 20 111m, a~ld for some purpose it is closed to 10 nun. Ihe ',p()slIrt' 11I1l~ must be 1l1creased four times. Opening it to 30 111111. flOl,lI 0111111. reduces the exposure to 4/9 of the standardized time. ~'()r I()\\' ~.)o\V.e,r ~v(~rk with .tnl~sl~litted.light, t1~eiris diaphrnglll in Ilu ph()t(.)gl.lphl lens (assull1l1lg It ISequipped With one) should not h~'.llIa~t'I'I:t1.ly. closed: .\Vhel1 not forcing rhe lens 10 its limit in 111:1 r 11I(lt':l1 ion, 11ISpel'llllss.lhlc to close the diaphraglll slightly, as a paJ'l!;" conr I'()I Oil exposure t 11 lit's. F()I' t'\t l't'lIldy 1:11' J'C objccts c.g., whole brain St'Clions ","it'" IIIIISIhe photo rl'aphed by trunsmiucd lig-hl, the Il,i<:rost'ope is omiu xl

et

and the lenses are mounted directly on the camera lens board. Then some means must be available for supporting the ,object and illumiriating it uniformly. An opal or ground glass placed some di~tance behind the object and suitably illuminated' from the rear will suffice. Under this condition, filters must be attached to the front of the photographic lens, as in ordinary camera work, and the filters must be of such optical quality that they do not affect the performance of the lens. With these exceptions, low-power photomicrography with transmitted light does not offer problems in technique radically different from.higher-power work.
I

Photomicrography

with Incident Light

The photography of small opaque objects at either low or high magnification by means of incident (often called indirect, or reflected) light has more in common with ordinary photography than has transmitted-light photomicrography. . Two. types of lighting are employe~ for opaque objects. In on' type, the light is projected from the sideagainst a reflector located back of the objective. This reflector turns ~he ~ourse ~f the rays alol~ r the optic axis into the back lens. Of the o~Jectlve, which ?ecoll1 .~, III effect,a condenser, concentraung the lighr on the object. Such illumination is known as vertical, or specular, illumination. It ~s cmplayed especially for metallurgical work, from low to th~ very highest maznifications. This application of it will be discussed m Chapter 5. . Vertical illumination is of little practical use in other lines. of photomicrography, because it does not cast shadows. Everything therefore is shown in flat lighting, devoid of contrast, except :vhere str~:mg differences in the absorption of light are presen~ in :rano~ls p~mlOns of an object. Those attempting to employ Vert1Callll~lmma:lOn f?r general photography of opaque objects invariably are disappointed m the results they achieve. . The other form of incident lighting is usua.lly caped obl~q~le top illumination. With ordinary photomicrographic eqUlpment, It ISconfined to objectives possessing' a considerable working ~istance be- ' twccn the front lens and the. object, so that a beam of lIght Cal: be projected at an angle, past the objective, to the surface of the object. For this reason, it is largely limited to medium- ~nd 100~-power work Magnification lip to 5~) diameters can be obtained WIth short-focus

180

THE TECHNIQUE

OF PHOTOMICROGRAPHY

PHOTOMICROGRAPHY

WITH

INCIDENT

LIGHT

181

microphotographic lenses, and this includes most of the, opaque work that is done. For the range between' 50X and roox, low-power objectives and' high-power oculars can be used successfully, if, suitable means of illumination are available. . When the distance between the front lens and the object is short, illumination by a single beam of light, becomes impractical because the angle of incidence is so great that excessive shadows are cast an.d only high spots on the object are illuminated. To obviate this condition various types of illuminators have been developed. They all are based upon the principle of illuminating the object by means of a cone of light more or less surrounding the objective. Among the more modern pieces of equipment for this purpose are th~ Lei~z ~ltropak and the Zeiss Epi- W condenser. These employ special objectives and an illuminating system which projects the light around the outside of the objective proper, but inside of an auxiliary shell, directly focussed by reflecting surfaces onto the object. With these devices, highpower views of.opaque objects may.be m.ade. Plate'4~ illustrates what can be accomplished even at a magnification of rooo diameters, One of the commonest problems of photography by indirect illumination lies in securing a considerable depth of focus. Most opaqu~ objects are very uneven in their surface contours and it is necessary to define sharply the low portions, as well as the high parts. This i,s,a case where stopping down the lens aperture by means of the ins diaphragm produces the desired result. The problem of obtaining just the proper amount of high lights and shadows with microscopic objects is analogous to the same problem with gross objects, p~)ftraits, etc. in ordinary photography, , Rotation of the object on the stage; changmg the angle of the lIght beam; use of a second supplemental light; diffusion of a straight beam by means of a groU1:d glass interposed; ,re-~eflecting a portion of the lizht back on the object from the OpposIte SIde, are some of the mcth ods which should be tried to obtain the desired result. In general, when the image looks good on the ground glass, it should make a good picture. It is well to remember, however, rhn: contrasts between light and shadow are apt to be ac .cnruat 'd in a picture, so that it is usually bet.ter to work on the Rat side, visu . ally, in order that the final results WIll not posscss an undue :1111011111 oj contrast. No one type of illumination will rake care of all opaque subjects, al

all possible magnifications. Three general systems will be found useful under different conditions. These are: (I) Unbalanced diffused light. This is secured by the use of from two to several tungsten lights of an Intensity suitabl~ to, the~ p~oximity to the object and the extent of magnificanon., Thl~ lIghnng IS u.seful only in the low magnification ranges ar:d .WIt~ falrl),' larg~ objects. The unbalance is secured either by a variation m the mtenslty of the lamps on opposite sides, or in the distances of the ~amps from th~ object. This form of photomicrography does not differ from ordinary
I~ 0;:::

'11~

FIG.

I14.

SPOT LIGI~

ILLUMI ATION, WITH ARC LA 11?

commercial and portrait photography, except that enlarged instead of reduced images are obtained.. . (2) Spot lighting with light from a smgle .source. T!11S tpe (.If illumination is especially valuable for slightly hl~her magl11ficat1~lI1s 11\ . cases where there is ample room to project the lI~ht alo!1g t!lC mic of the objective. The method is shown diagrammatically m FIgurc I ~+ For the higher powers, which require an intense light, an arc lamp with a focussing condenser is essential. Such a lamp, ?10unted on. a. (lo(~r stand (Figure 53) which can be adjusted as to height and POSItion, IS idcal. For very delicate objects, which might be dan:aged by. heat, it will be nccessary in addition to mount ~ w~ter. cooling ccll. 111 the beam; near the lamp. This method of illumination ISalso useful III lowpowcr work where objects of large area and little surfacc. r~~i f, :1I:~ involved. Rather than employ an ~rc lamp here, however, It ISh~tt<:1 t!) ch,lIlgc to ,1 tungsten lamp as a high-}nt?l1sitf arc is not rC~lulr~'d; ncit her is <1 [ocusscd h <1111. One essential 111 rhis method of Itghtll1 r is I Ill' S 'ClIl'in~'of even illuminnrion ()V 'I' rh' .nrir area. 1 his is nccom plisIlvd I>y p'I:lcil1g' the l.unp .sllfTlci'nlly fur :lw:\y to r~dll~(.' the dif 'I' 'lilT ill t h ' It'll rths of t Ill' It rht pat hs to the t wn opposite Sides of the

182

THE

TECHl

IQUE

OF PHOTOMT.CROGRAPHY

PHOTOMICROGRAPHY

WI'I'II

I <:(1)10: T 1.((;(('1'

object to a small value. Figures I 15 A and B illustrate the reason for this. At A the relative lengths of the paths (P and P,) of the rays are to each other as 3: 4. The intensity of the illumination varies inversely as the square of the distance, and hence one side ,of the object isillurninated by 16 units of light as against the 9 units which the distant

~f s~vera~ ways. Figures I 16A and H illustrate some of the possihili ties 1I1 this method. In A, 711. is a small mirror, plain or concave, :IS ~equired? which reflects the light, after passing the object, bacl ont 0 It at a suitable angle. This may be used alonc or in combination wit h

Object Object

Axis of Microscope

,A

Object

FIG. 115.

Method of Securing Even illumination

on Both Sides of Picture

side receives. This is nearly a two to one ratio, and an uneven picture is sure to result. At B the paths are to each other as 10: 11, the light intensity relationship is as 100: 121 (i.e., as 5: 6). This difference ordinarily is not sufficient to cause an objectionable variation in the intensity on the finished print. (3) Side illumination, deflected onto the object. This method is particularly applicable to higher magnifications, where the working distance between the object and lens is at a minimum, although it can be used for low-power work as well, if the necessary reflectors arc available. . For high-power work an arc lamp with a focussing condcns r is employed to project a beam at right angles to the opri axis, b .tw 'en the lens and object. This beam is deflected to the obj et in any on'

I
B
FIG. 116

n li duly frosted ground glass (g) which diffuses the light and softcns IIll' shadows. A n opposite arrangement is shown in Figure I I (iH. l' is 11 smull C)oo prism located in the path of the rays, deflecting thCIII down OIl till' ohjccr, When counter illumination is desired with rhis :11' 1.111 rt'IIIt'lIt, the size of the light beam must be such that not all of it p.t Sl'Sthrough the prism. That which does not is reflected [rom till' LtI' side, preferably by means of a dead white surfn .c, hucl to the hinl.. \ Vit h t hcse gcn<.:r:1\sugg'stions before him, Ihe 11rat'tira\ I hOlollli('l'o rraphel' r:11Idevise 11IeanSto uuvt :111 problem. "01' low

THE

TECHNIQUE

OF PHOTOMICROGRAPHY

COMMON

FAULTS IN PHOTOMICROGRAPHY

power work, when this system is employed, mirrors or reflectors of a size commensurate with, the area to be covered must be used. The light beam itself should also be of large di,am~ter. , . ' One occasionally has to photograph, by incident light, objects which have strongly reflecting surfaces. These cause objectionable high lights in the picture. As reflected light is always more or le~s polarized, these reflections can be subdued and even completely eliminated by the use of polarizing films such as Polaroid. Ordinarily one such film, mounted over the front of the lens and rotated to the position of extinction for the reflected rays, will be ample. For severe cases it may be desirable to employ a polarizer in the path of the illuminating rays, also. Any effect desired may be obtained by rotating both prisms until the combination is satisfactory. , It is only occasionally that filters other than yellow or ?range a:-e used with opaque objects. Whatever is required can be inserted m the light train, if the filter size is sufficient. Otherwise they can be mounted on the front of the lens. . It is hardly necessary to add that the time of ~xposu~e with op~que illumination is greatly increased over that reqUlre~ With transl11l.tted light at similar magnifications. The best method, If much work IS to be done along this line, is to make test exposures, Just as recommended for transmitted light. Common Faults in Pbotomicrograpby What are the faults most likely to be detected in the work of the beginner in photomicrography? An answer to this question may be of material aid in helping to eliminate them. Although the faults should be self-evident from the information given covering methods for the production of high-quality pictures, an analysis of them may put the emphasis where it is most needed. , . We can divide the causes of low-quality work into the following groups: . _ . ( I) Faults attributable to poor apparatus, poor optical eqlllpment, and poor focussing: , One should not be hasty in ascribing failures to the qualIty.of the equipment, but the possibility sho~:lld not be overlooked. Pictures falling into this class are charactenzed by a lack of sharpness, even when the magnification is well within the range where no 'mpty Ill:lg nification should be present. It may be due to poor fOCllSSlI1g only,

It is impossible to 'focus accurately on the ground glass without using some form of magnifier. This is the first condition to be suspected if non-sharp pictures are encountere.d. If the trouble lies in the equipment, the actual cause may be anyone of several conditions, each of which should be carefully checked as the possible primary or contributory factor. Vibration, instability of the fine-focus mechanism during exposure, or poor quality of objective are all possible. When achromatic objectives are employed, a possible cause can be introduced by previously focussing with a filter of one color (or no filter at all), followed by the introduction ofa filter of a different color without rcfocussing. In such cases of this kind, the answer can be found by a process of elimination. Any of these troubles, when finally located, can be clim inatcd in some manner, though not always without expense. _(2) Faults due to improper exposures or development: . When this type of fault is present, it indicates a need for th be rill ncr to become better educated in the strictly photographic tc .hnique involved. Outside advice from some friend who possesses experience in photography will be a bIg help here. Pictures coming in this group are either flat, lacking cant rast , l'\TIl when the object was well stained and well differentiated, or the\' ;lll' too contrasty and require a long time to print because the ncgatlH' is , rrrcmcly dense. _ One can have a correct .exposure, either under- or overd vclopcd, - 01' correct development of an under- or overexposed negative, ln ud dit ion, four other combination conditions are possible: i.e., (I) under , posurc, underdevelopment; (2) underexposure, overdev lopmcnt , ( i) overexposure, underdevelopment; and (4) overexposure, overdo
ilopmcnt.

If one must work alone, without advice from someone qualified to ive it, the place to begin to correct the trouble is with the de vc lop III -nr. By using the specific developer recommended by the IlHlI1l1f:ll' IlInT of the plates, at the correct temperature, for the COlT .spondin r Iiuu- r '('ollllllcndcd for normal contrast, the development fact or ca 11 hc csrnhlishcd. Such dev lopmcnt of a test exposure, as outlined pn' iousl)', should provide a standard for future guidan .c, Th Tl' is, however, a simpl visual tcst which can often he ~lppli.d \0 a Ilq~:ltivl" rhnt will illdit':lIl' proper, over, or underexposure, provided till' de 101l111'I\t is substauriully COl'!' .cr.

186

THE

TECHNIQUE

OF PHOTO lICROGRAPHY

COMMON

FAULTS lN PHOTOMICROGRAPHY

An underexposed plate held in such manner that a poorly illuminated part of the room is back of it, and with a bright light from a window falling on the top surface, will show a positive image with the film side up (i.e., toward the light and the viewer), but will not show such a positive image when the glass side is up. . An overexposed plate is just the reverse; a positive image is seen when the glass side is up, but the film side shows black. A properly exposed plate should show a very feeble positive image from both sides. (3) Faults due to an improper color filter: The general tendency in the use of filters is to produce too much contrast. Parts of the object which ought to reveal considerable structure are portrayed absolutely black in tbe print. Often the photon:icrograpber himself is disappointed in the final results, The neganve may look wonderful, with plenty of detail everywhere, but it. seems impossible to make a print that looks like the negative. When the detail is in the shadows, the high lights are washed out; printing until the latter come out properly makes the darker .portions black. The' best that can be done under these circumstances is to use the softest available paper (#0 or #00) and a very soft developer. In future cases, the place to correct the trouble is at the color filter. One should be chosen which does not make any portion of the object appear dark and lacking in detail. The proper contrast is that with which, using normal (# 2) paper, the very darkest detail in the object will just reach blackness in the development of the print when the whitest portion (possibly the clear background) is just starting to print. In this way the entire scale of the paper, from pure white to jet black, is utilized. There is one limiting condition, in this respect, for which there is no complete remedy. This occurs when an unusually dark object is to be photographed against a clear background. The filter must be chosen with reference to a proper portrayal of the object and the exposure must be ample fully to record the object on the plate. This means a gross overexposure of the clear background, which cannot, be equalized, in all cases, by underdevelopment. The final result is a properly printed image of the object on a perfectly white background. This is by no means objectionable, however. As a matter of fact, some workers strive for a white background for every picture, (4) Faults due to improper illumination: Here we may place blame for the majority of poor r sulrs in photomicrography. Failure to obtain critical lighting, improp 'J" ('(.'111 .rin T

of the .illumination system, the introduction of diffraction effects, and other conditions of similar nature, all detractfrom ideal results. When critical lighting is not employed, a very common result is an uneven illumination of the field. The result is a picture such as that

C
(:l1l1s(.d

.)

~'

FIG. 117.' U EVE TLLUMINATIO hy rhc failure to place 'condenser in the proper position 11If{, wit h :\ resultant vignetting cffccr produced by the diaphragms 111('uI ion wh 'I" they can function properly.

for crit i .nl li~hl not h 'il1~ in t lu-

110\\'11

ill Fi

TUr<: 117,

"Illn.alld

]lniphery,

as S 'CI1on

The variation in rhc inr nsity the rround rl:1SS,

i uully, hilt the plate shows it.

the light, :11 tilt' not he evident The cnusc of the dark border around
of

Illay

188

THE TECI-INIQtJE OF PHOTOMICROGRAPHY

COMMON

FAULTS IN PHOTOMICRQGRAPHY

lily

an otherwise good picture is generally to be found in the vignetting effect of either the aperture or field diaphragm when they are not at the exact place in the system to perform their functions properly. With critical lighting - both diaphragms in their proper position - a

position, the vignetting effect results. The substage diaphragm, which is the aperture diaphragm, is far more susceptible to a slight chang . ill position than is the field diaphragm; hence, as the substage condenser

FIG. IIS.

LIGHT! G

OT A}"'1AL
FHI.

in combination with lack of alignment of the light with the optic axis. A common fault when a mirror must be used, as with a vertical camera.

A condition. caused by lack of critical illumination

Ill).

1':XCu:.SIVE DIFFRACTION

CAUSED BY A NARROW

CQNE

01'

TLI. MINA'IION

vignetting effect is impossible. If the aperture diaphragm be closed down, it is manifested only as a darkening of the lighting over the entire area, but the lighting remains uniform. When the field diaphragm is closed, there should be no semi-shadow cast, but a sharp image of the edge of the diaphragm should circumscribe the dinrn t r of the picture. When either of these diaphragms does not oc lip its proper

All- I he dnrl er lcmcnts in the tis. ue arc seen to be surrounded by a white hurder. 111I'1'1'('1 Io 'us is almost impossible under this condition, as that whi h nppl':II'h 10 1(11 I' till' IIIOSI' striking illlage on the grOWl I glass is not the proper onc, as cun IH' SI'I'Il III Ihi pkllll'C. -

i removed
till'

result
1,:Il'I,

from its proper position,'carrying rh diaphragm with it, be an uneven illumination ;IS shown in Figure I 17 of ccnt rulii v of the illumination Systl'lIl, combined with t hr
11I:IY

111 '11 .c

of crit iC:J1 'illlllllillatioll,

is :1 vt'r~: couuuon fault,

especially

THE TECH

IQUE OF PHOTOMICROGRAPHY

COMMON

FAULTS 1

PHOTOMICROGRAl'HY

when vertical cameras are used. An improper setting of the mirror results in decentering the light. The resultant picture appears as in Figure I 18. It is hard to detect on the ground glass visually. However, w~en t~e:fiel~ diaphragr? is sharply focussed on the plane of'the object, ~s III critical lIghtIllg, this cannot occur, for the edge of the diaphragm ISseen immediately any portion of it falls within the picture area. Another common fault is the introduction of diffraction effects in photographing objects where diffraction is unnecessary to reveal the str~cture. Diffra~tion is necessary with unstained transparent objects, as Illustrated by FIgure I 12, but when introduced in stained histological se.ctions, t~e e~ect is very unsatisfactory. The resulting appearance ISshown III FIgure I 19. Attempts to use objectives without eyepieces, or microphotographic lense~ in a smaJ~-tube microscop~, usually produces a fogged area on the pIcture, which results from internal reflections from the polished brass sleeve into .whicl~ the ey~piece fits. The fogged spot may take the form o~ a sol.Id white ~rea m the center of the print or a light ring' of larger dimension. Its SIze and shape are determined by the projecnon dI~t~nce. The ring form is illustrated in Figure I20. Insert.lOn of a black paper tube or a metal sleeve containing an int~rnal diaphragm of a diameter smaller than that of the eyepiece p9rnon of the tube, will correct this trouble. (s) Faulty pictures due to improper magnification: The. particular faults in this group are not technical but, rather, aesthetic ones. The deviation from ideal results is largely confined to objects which are entities and should either be shown complete or only a small portion at high power. For instance, it is an unsatisfactory picture which shows an amoeba entire, except for the end of one of the pseudopodia, which projects outside the picture. The condition is analogous to amateur snapshots, where the feet (or top of the head) of an individual are missing. From the artistic standpoint one is as bad as th~ other. A sligh~ reduction in the magnification would have sufficed to include everythmg. Unfortunately, many pictures result froI? causes bey?n? t~e co.ntrol of the operator. The range of optical eqUlpn:er~t a?d limitations III camera extension are responsible. Where these limitations are not present, however, one should strive to make his pictures aesthetically pleasing. (6). Unevenness of background traceable to plate emulsion: . Th~s ~annot be classed as a preventable fault, in photomicrography, since It ISlargely beyond the control of the operator. It is included in

this list, however, as it may appear to be a d~fect in technique and cause many a headache in attempts to elimi~ate It. . . This sort of unevenness of background IS somewhat similar to Ihe effect resulting from non-alignment of the light, although not so con-

FIG. 120.

FLARE RING FOGGI G

fogged circle, concentric with rhe imagc, produced by internal reflect ion fm1l\ Ihi' microscope tube, when no eyepiece is employed and the light can proJ~'ct Oil till' hili ~ t uhc. Depending on the projection distance, the fogged area can CH her \i(' :I 11111( Ill' a solid central spot.

pit.'1I01lS. It will not be noticed on a negative.' but, ~s a print is ~\cvd Clp'd, the masked circle shows up .at o.ne Side bcfor~ t1;cre IS :111)' indication of printing on the OppOSIte sIUe. c()l1lbl1::1rtol1. of I\~o -oudirions is required to produce the effect. The first IS <1. sltg:llI dif f -rcnc - in the thickness of the emulsion of the plate, which IS v 'I')' nnunon though not necessarily uniyersal: . Supcrimpos .d on litis lIll Vt'lllll'SSof Ihe .mulsion must be a condit inn wh 're till' CXpOSUI'l' of till' (':H'I, rround is so full as to affecl i hc silv .r cnrirclv thl'OII.\1 tit!' -mul ion. As pl'l'viously poil1H'(,1 ()III, i his IllW',tI>l'dOlll' dcliln-r.n cl . ill 1111111 CIISt'S, ill order 11t:11 t hc Ol>llTI Ill:1y 1'l'l'l'I\'(' :ullpk njlosu\'t'. , As

THE

TECH

IQUE OF PHOTOMICROGRAPHY

IMAGING

OF LIGHT

SOURCE VERSUS LIGHT CONDENSER

there is ~ore silver in the thick portion of the emulsion, it builds up a blacker Image and the final print is lighter on that side. than on the side where. there is less silver. Although this detracts from an ideal picture, it cannot be helped: It never occurs with sections and objects which fill the entire field. Practical Advantages Source of Critical Illumination

by lmaging the Light

~n discussing the optical principles of photomicrography in Chapter It was pointed out that critical illumination for medium- and highpower work can be secured by eith r imaging the light source or imaging- the light condenser on the plane of the object. The latter method is employed in practically every complete commercial outfit sold in this country. The reason is obvious: it yields a uniformly illuminated field, regardless of the nature of the light source: For instance, a concentrated-filament tpngst.en lamp gives just as perfect lighting as the most uniform light source, whereas in the method of imaging the light source on the plane of the object one sees every coil of the filament, superimposed on the object. Because of this, it would appear that there is nothing to be said in favor of the alternate method. So far it has been assumed that the standard setup, using the light condenser as the light source, is employed. This has been done in order to present the practical aspects of photomicrography without cornplicating the problem in the mind of the beginner. Before leaving the subject of the technical aspects of photomicrography, however, there is something to be said in favor of the alternate method of obtaining critical illumination. The standard method (often called the Kohler method) is, in spite of its beautiful uniform illumination, open to several objections. With a view to obviating these, the author experimented many years ago with a modified setup, utilizing the principle of imaging the light source instead of the light condenser. It has been so satisfactory that it has been used in fully 95 per cent of his work, for the past twenty years. When a situation arises, as happens occasionally, where the so-called Kohler method will prove advantageous, a changeover can be effected within a few seconds. A comparison of the advantages and limitations of the two methods
I,

may be helpful to many who have run into complications which Iht,y have not been successful in overcoming. _ Experience has demonstrated that rapid exposures are not desirable for ordinary photomicrographic work. It is much simpler to work ill the range of I to 60 seconds than in fractional-second exposures. Further, an intense light is objectionable not only because it rends to fade many delicate stains and some gelatin filters, but because it is accompanied by excessive heat. Heat not only can damage a slid, or an objective; it can 'also cause a change in focus due to expansion of various parts during exposure. Both of these conditions are a. factor when light from an int .nsc source is imaged on the plane of the object either directly or as the condenser image. On the other hand, substitution of a lowcr-inrcnsit light without further alteration in the setup does not give equivalent results photographically, as commercial low-intensity lamps ar ddi cicnt in blue rays. , ' , It is because of the heat effects that many commercial outfits inc-lude l'ooling cells, even with tungsten light illumination. 'I he light source in the arrangement ordinarily employ d I> the author is a 5oo-watt Ostram gas-filled tungsten lamp (rated J wut t Pl'l -nndle) .. But this is not used as the effective light source, for dirccrlv in front of it are placed two finely frosted glass plates, the polixlu-d surfaces together. This provides a double diffusion and absolute lI11i formity over a circular area two inches in diameter. This disk of li rht -onsritutes the actual light. source. It is located about one met Cl' dis runr from the microscope .. Directly in front of the frosted glass disks is located an iris diaphragm sufficiently large to utilize the entire Iwo ill .h circle of light, should it be necessary to do so. This diaphru fill -onstitutcs the field diaphragm. The field condenser is located Oil IIll' optical bench so that the diaphragm lies in its principal focal plane, and thus the rays to the microscope are approximately parallel. The urc inter .cptcd by an auxiliary centering condenser of long focus (70 cm.) which converges them slightly into the substage condenser. From rhe operating standpoint there is no difference in the prol'l' durc followed in eff cting 'critical lighting, as described earlier ill till' -huprcr. J\ ftcr foclIssingtthe objective on an object', Ihe condenser is [n -usscd until an image of the ficld diaphragm is sharply ddilll'd 011 tIll' plane of the ohj 'Cl, the diaphra 'l1l I>l'illg closed slIfiil'ielllly to:tl luw this 10 he done, The apl'1'tlll'l' of the suhstn 1'(' l'()lldl'llsl'r is dc
I l

194

THE TECHNIQUE

OF PHOTOMICROGRAPHY

IMAGING

OF LIGHT

SOURCE VERSUS LIGHT CONDENSER

195

terrnined in the usual manner by removing the eyepiece and observing the back lens of the objective. . , The majority of the illustrative micrographs in Chapter 8 were taken with this lighting arrangement. The question naturally arises, What are its advantages over the so-called Kohler method?" , . There are several. In tl:e first place, no cooling cell is required. 'A simple test, made by placmg the bulb of a chemical thermometer at the focal point of the substage condenser, with the diaphragm of the latter open, showed a rise of but one degree Centigrade in fi ve minutes. Removing the frosted glass and changing over to the Kohler method, with the same lamp, a rise of five degrees in three minutes occurred. This indicates that the heat is reduced to about one-eighth, a far greater reduction than can be obtained with a cooling cell. . In the second place, the exposure time is lengthened to' a point where Ideal exposu,res can be made and calculated exposures carried out to a small fractional percentage of error. That the frosted glass has little effect on the nature of the light, so far as altering filter values is concerned, is shown by the transmission curve, of the two glasses (Figure 12 I). For comparison, a single piece of opal glass 'was also graphed on the same sheet. This shows that opal glass could be substituted for the frosted glass, both having nearly a straight-line transmission of between 40 per cent and 50 per cent. Either of these provides the one element ordinarily lacking to make criticallightil1O' by irnacing the light source, practical - i.e., a large area of absolutely unifor~ intensity over the entire surface. Though the exposure is practically doubled by the use of the glass, the heat is reduced many times more than the light intensity, giving an approximation of that ideal condition, " cold light." Some workers feel that equivalent results can be obtained by the insertion of n~utral-tint filters. Bausch & Lomb supply a set of neutral fil~e~sfor this purpose, but a glance at Figure 122, showing the transmISSIOncurves of these filters, reveals that they fail to zive such a straight-line reduction in light intensity as the name might imply. On account of the small heat effect present with the author's method, e~posures can run into many minutes, without the focus altering in the sl~ghtest. For extrem~ conditions where the filter factor may run very hIgh and an exposure m hours would be required, it is a simple matter to ren?ove the frosted glass, switch the position of the lamp condenser and diaphragm to the alternate method; then, if this is nor sufficient, remove the lamp and replace it with an arc lamp and a cooling cell.

In this way maximum flexibility is secured. It should be noted. that the heat problem is automatically taken care, of, so far as the SpeCIl1l'11 and objective are concerned, by the u.se of fil~ers .of extr~mely low transmission characteristics. The cooling cell IS still required, however, when an arc is used, as a protection for the filters which call
100
I

100 90

90

Il'i

u re L int Filters

( S (!C

80

80

o \ o \
A
2 0 ie .es, F~osi d Gl 'is
1"-

I1 11I1
t50%
t/'

0J::.- I30

l...

j...-

I- I-

'--

......

1\ 1\

o \
0

:-50 %of~ /"

V I

Oal Glass

B'

I1 'I
r--

20

, ,
0 0

0"

~~\1/
I-"
\

JI~

o \
0

50 %ifY- 1-"""t-t--

I
150%

./

I-.

--

/V
j

r c

~~~OOM~~~OOM~~~OOM~ Wavelength in Millimicrons

~~~OOM~~~OOM_~~OOOO~
Wavelength in Milli'mict'outf

FIG.

121. Transmission Curves of Frosted and Opal Glass.

FIG. 122. Transmission ClIJ'\'l" (lr B. & L. Set of cut rnl Fihl'l'\

nsily be damaged by the extra heat which they would otherwise ahsorb. . \ Vhere an optical bench is not provided in the outfit it ~l1a y ~), :I,III t lc morc of a problem to introduce.the author's.method of III1Imlllal\()I~, hut wherever it is possible to use It, results will be found to be gmll fying. '

METALLOGRAPHY

197

Chapter 5
SPECIAL PHOTOMICROGRAPHIC PROCESSES

Some types of photomicrographic work are of interest only to those engaged in specific or limited fields of research. While the general photomicrograph problems involved in these do not differ from those of ordinary lines of microscopy, in the majority of cases the work must be accomplished with specialized equipment, or at least' with the help of auxiliary apparatus not required for ordinary purposes. In order to avoid complicating matters for the general microscopist not concerned with these specialized techniques, they have been largely eliminated from consideration in Chapter 4. Some of these find very extensive application in their respective fields and 'the volume of work turned out entitles them to rank among the leaders in the photomicrographic field. Others are at present only of limited value, but advancing rapidly as their value to science and industry are being recognized. First on the list, in point of importance, can be placed the photomicrography of metals. : ,
METALLOGRAPHY

Metallography, the science of studying, interpreting, and photographing the physical structure of metals by means of the microscope requires a radically different type of equipment from that employed for ordinary photomicrographic work. In the first place, the objects to be examined and photographed are entirely in the opaque class, and hence incident light alone must be employed. Although low-power work on metals can be accomplished satisfactorily by means of the various types of oblique illumination described in Chapter 4, by far . the greater part of metallographic problems must be solved by recourse to high magnifications far beyond those possible with the methods available up until very recent years. The fundamental optics of rnetallography do not differ from those already discussed as applying to photomicrography as a whole. High-aperture objectives are J'e-

quired for high resolution, ,the illumin~tion ~ssubject to the same laws, as regards intensity 111 relation to magnification, etc., and hence a powerful light source, properly focusscd ~n the surf~ce of the speCImen, is essential. Yet this must be accomplished even 111 the case of an objective which may have a working distance of but a few thousandths of an inch between its front lens and the metal surface. The only solution lies in the employment of a .vertical illumin~tor located above the objective, so designed as to r~cel~Tea powerfl~l hgl~t beam from the side, project it through the obJectlve'onto the speCImen: and allow the image picked up by the objective to pass it to form the image. There are several different methods of effecting this, so far as the design of the illuminator is concerned. The essential elementin the illuminator is a reflecting surface which must lie in a 45 posirionabove the back lens of the objective. This reflector cas be a 45 prism, located so that it just covers one-half of the FIG, 123. Bausch and Lomb Vertical 11111111111.1 area of the back lens of the tor with Lamp attached objective, a semicircular mir. . . ror in the same position, double 45 pnsms cemented together \VJI h half silvering on one prism, or a thin plain glass reflector. Th~ latter is the commonly preferred form, f?r sev.eral.reasons, among \~/h\(:h.arc the reduced aperture, non-centralized hght111g, and uneven illumina don present in the prism illuminators. Figure 123 shows the Bausch &: I.omb vertical illuminator with lamp attached. Two types of metallographic outfits. are employed. That l.lI0re -loscly associated with ordinary phot?mlc~ography empl?ys a Jl1I<. . O 'ope differing from a conventional blOloglcal.stand only 111 p'0SSCSS\l~ r a srngc which can be adjusted up and down 111 th.e optic a IS, and 1.11 h ;lIg equipped with a vertical illul1lil~at.or. sln~plc .stand of this 1\ pc, of Bausch 'L~mb. \llanll.LlCtllr " I~ Illustrated 111 \'Igurc I +. It .1Il he used in combination \\'1\ h :I vert 1(':11 camera, the hc.uu of h rht hl'illg projectcd into till' ilhuuinntur illStl';ul 0.r.:lg:lil1~tth '. IlIil'l:or, :IS virh t musiuiucd light. It is (H.'C:lIIS'the \lCISItIOI1 of the illuminator
0 0 0

'9

SPECIAL PHOTOMICROGRAPHIC

PROCESSES

METALLOGRAPHY

199

cannot vary with respect to the position of the light beam, that the stage adjustment is essential. Instruments of this type can also be used in the horizontal position with large horizontal cameras. When this is done, the optical bench must lie at .right angles to the camera axis. Where both metallographic and transmitted-light photography are to be done, two optical benches are required. The setup under this condition is shown diagrammat ically in Figure 125.

,/' Right-angle

optical bench

[IIOO~
Camera

I=#=::!:=====itl
St,.,1Iight optical bench

ILam 1>

1'1(;. 125.

Combined

Straight

and Right-Angle , Camera

Optical

Benches, HorizOII1:l1 I):"

FIG.

124.

Bausch and Lomb Simple Metallurgical

'I icroscopc

For more pretentious metallographic work, especially where the in 'rest lies solely in the realm of metals, the Le Chatelier design of out fir, often called the inverted type, is in general use. The Bausch '. f .omb metalloscope, type ILS, which is of this form, is shown in Fig urc 126. The surface of the metal specimen to be examined is placed Oil the stage face down and is illuminated and examined from below. Ihe path of light through this instrument is outlined in Figu re 1 7. f hat portion of the apparatus which comprises the microscope proper i illustrated in Figure 128, with the various parts designated. It is beyond the scope of this volume to enter into the technique of reparation of the metal samples, which includes polishing and et .hin r. \ t is ne .dlcss to add that no.photornicrographscan be produc .d, 110 uuurcr how perfect the technique of taking them, that are het! 'I' t 11:t-1l IIt, samples th rnsclvcs. It follows, therefore, that the highest qunlit y \ urkmunship in rhe preparation of d,1 samples must h - assumed ill dis 'us, in r t hc strictly phoromicroprnphic:ISpt't'ts involv xl. F()I' :\11 OIH' itllt'ITstt-d ill t:lkillg lip this line of work, ",host' sole previous npt-ri 11 " has hecn nlon r gellerallllil'l'o~t'opi -:tllill<.~,it tll:ty be well to poillt
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200

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES

METALLOGRAPHY

201

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METALLOGRAPHY

203

out a few places where considerable divergence occurs in the optics of, the two conditions. Metals differ from ordinary microscopical objects in that no cover glass is employed. As the cover glass is apart of the optical system of the objective, omission of the cover glass makes it obligatory to employ an entirely different series of objectives, especially designed to work without cover glasses. This does not apply to homogeneous immer- , sion lenses which will work equally well in either case, so far as presence or omission of the cover glass is concerned, but even here other conditions enter to make a distinction between the lenses required. These are: first, the desirability of having the lenses of the objective located as close as possible to the vertical illuminator, in metallographic work, in order to reduce reflections; and second, the employment of a longer tube length in metallography. , The appearance of a metal specimen is entirely different under vertical illumination from that under oblique lighting. The reason for this may be understood if we compare what happens in the case of a highly polished surface when illuminated by the two methods. Such a surface behaves as a mirror, so that if we are observing it under a microscope with oblique light falling upon it, according to the law that the angle of incidence is equal to the angle of reflection, the total light would be reflected on the side of the microscope opposite to that from which it is illuminated. No light would be reflected into the microscope, and the specimen would appear dark. But with verticalillumination, all light is reflected directly back into the microscope and a dazzling white surface results. In other words, the two appearances are exactly opposite. It is for this reason that vertical illumination, which must be employed with high powers, is adopted for low-power work also. Interpretation of micrographs can thus follow a consistent system, regardless of magnification. When for some reason it is found desirable to utilize a different type of illumination for a polished and etched metal specimen, in taking a photomicrograph, the method should always be stated, as otherwise even an expert metallurgist might not be sure as to whether the effect was obtained by illumination or by some unusual etch, since varying the etchant can also result in similar reversals in appearance. Although the fundamental optics applying to metallographic work are not essentially different from those of general photomicrography, it is not so apparent, with unit outfits such as the Bausch & Lomb Metalloscope, just what is involved, for everything has been so designed that proper opri .al conditions are met .. One has only to follow the operating instructions furnished with

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204

SPECIAL PHOTOMICROGRAPHIC

PROCESSES

DARK FIELD PHOTOMICROGRAPHY

the .outfit. Nevertheless, one is aided to proper understanding of the eqmpment by becoming familiar with the theoretical principles of illumination underlying the practical setup. . When working with the conventional form of microscope equipped with a vertical illuminator, the need for a thorough knowledge .of the principles of illumination is greater, for in this case the entire arrangement. of the light train is left to the ingenuity of the individual. !t ISpre~erable in metallographic work to utilize the method of imagm~ the light condenser on the plane of the object. This means that t~e lIght s.ourc~ ~ust be focussed on the rear focal plane of the objectrve. ~hlS posltlon varies somewhat with different objectives, but is approximately at the opening into the vertical illuminator, or the diaphragm of the latter when it is equipped with one. When the light is Image~ on. the back focal plane of the objective, the rays pass from the ob)e.ctlve to the metal surface substantially parallel, illuminating the maximum area which can be covered by the objective. l\Ily preferred method of accomplishing this illumination is to have a parallel beam from the lamp condenser, located at the distal end of the .optical bench~ enter a 20 cm. (focal length) condenser .which projects the beam mto the vertical illuminator. This condenser can be moved on ~he optica.l bench to adapt it to each objective, but the usual distance ISaround ItS focal length from the diaphragm of the illuminator. The !atter serves as the aperture diaphragm, while a second large diaphragm IS located between the illuminator and condenser (about 6 or 7 cm. from the latter). This serves as a field diaphragm, and being so near the microscope, is under control at all times. While exact critical illumination is not so imperative in the case of metallographic work as with transmitted light, the nearer it is approximated, the better will be the results. Care must be taken not to allow a larger cone of light to be projected into the illuminator than can be accommodated by the back lens of the objective, for it will only cause annoying internal reflections. With this system of illumination it is possible to block out the central portion of the beam by means of a disk III the center of the aperture diaphragm. This is called conical illumin~tio.n. It tends to i?troduce some shadow effects, especially when the disk ISdecentered slightly, or when a segment of the outside light cone is also suppressed. If achromatic objectives are to be used for rnctallographic work, superior results will be obtained by the use of a green filter, sin e this is the particular spectral band for which rh objectives are corrected. As arc lamps are used almost exclusively for l11l'taIIographic work on

account of the low efficiency of vertical illuminators of the plain glass type (as much as 90 per cent of the light may be lost), a high amount of ultra-violet is present. As plates are usually quite sensitive to short waves and as achromatic objectives are not well corrected in this IT Bion, it is desirable to eliminate all the shorter waves, or an inferior ill! age will result. Plates sensitive to green must be used in this case. Wratten Metallographic plates are especially suited. Wherever possible, apochromatic objectives should be employed, :IS blue filters can be employed with these and the resolution improved materially. Color-sensitized plates are not required with blue light.

FIG.

129.

Diaphragm

Stops for I?ark Field

DARK

FIELD

PHOTOMICROGRAPHY

. Dark field microscopy plays a very large and important role ill ccr rain specific lines of research, notably ~acteriology and colloidu I Cht'II1 isrry. It,Svalue i~ l:evealing ~xtremely n:iI~ute parti~les in suspension und particularly living, unstamed bacteria m a hanging-drop culture, is largely responsible for its frequent application in such lines, Oil t hc other hand, the photomicrographic aspect of dark field is of lesser im portance, This is due in part to the difficulties involved in tal ing rh ' extremely short exposures required for moving objects. A second ~'ea so;, is that little structural detail is evident in the average dark held micrograph, further, the diagnostic value ~f the moving parti71cs ,or organisms p~esent il: visual dark-field work ISlargel~ lost In a srill I)tt' rurc. For mstrucnon purposes, however, a definite need for tart field micrographs exists and a photomicrographer should he prepared to take such pIctureS when they are required. " ' From the strictly scientific standpoint, darkficld mlcr~)sc()py I~con filled c: .lusivcly to high-power work. The amateur nucros 'OIHst;,Oil the other hand, delights in the beautiful appearance of larger or ralll~IIIS when illuminated hrillinnrlv on a dart field, Low-power durl f1l'ld condensers, although nvailuhlc commercially, are not in t'Ollllll~ln I,I:W, nor nrc thcv ncccssnrv, hlT;IIISt':I ct'nl),;11stop, such as shown in 1'1' \II" I (), illtl'l'posl'd u'lIdt'I'I11':lth~IIIurdinurv suhst:l rt c-ondcuscr is :11> 'ffl'ct ivc as ;1s)ll't'i:lIIy dcsi rued ('(lI\d -n -r \\ o\lld he, 'I her , urc s iv

206

SPECIAL PHOTOMICROGRAPHIC'

PROCESSES

DARK FIELD PHOTOMICROGRAPHY

eral forms of high-power dark field condensers available. Their principle of operation is essentially the same as that employed for lowpower work with a central stop in combination with an' ordinary condenser. The two most commonly used for photomicrographic, work are the Paraboloid and the Cardioid (or bispherical). The former provides a wider latitude in operation, as the focal point is not so critically defined, while the latter giv.;es a more intense illumination, providing the condenser is accurately focussed on the plane of the o~~ jeer. The path of the rays in the Cardioid condenser is illustrated in Figure 130. The problems involved in dark field photomicrography are determined largely by the nature of the object. Where living organisms in motion are involved, or Brownian movement (pedesis) in colloidal particles is present, a fractional-second exposure is necessary.the exact time allowable depending on the speed of movement. As magnification amplifies the apparent speed, as well as the size of the object, keeping the magnification as low 'as possible is a help, since the intensity of the light varies inversely as the square of the magnification. This means that to reduce the magnification to one-half, the exposure is only one-quarter as long and the movement reduced proportionately. Fast plates are also desirable for this class of work. Enlarging the negative to the original size (i.e., two times) gives the desired object size FIG, 130. Path of Rays in the Cardioid Dark with only one-quarter the moField Condenser tion. Where motion is not present, and minute organisms must be shown as contrasty as possible, both the exposure and development of the negative should be on the long or contrasty side. On the other band, when a decided structure is present and can be demonstrated, the exposure should be sufficiently long to register detail in the darker portions, while the development must be on the soft, or less contrasty, side. It is better to use a contrast developer in the f rst case and a soft developer in the second, Sometimes ir pays to change plates as well.

Every attempt should be made to keep the field non-illuminated so it will appear black in the finished micrograph. It goes without say ing that only an arc lamp will give ideal results in high-power work. Low-power dark field micrographs, however, can be successfully made with a soo-watt tungsten lamp. Unless one is so experienced that he can tell the order of magnitudt of the exposure necessary under varying conditions, it is worth while to make a test strip exposure, at the start of this typ~ of work. . Photomicrography with the slit ultramicroscope is in the same CI:1SS as dark field work and subject to the same conditions in the photography of colloidal solutions. Very little of this type .of work is call 'd for, or justified. When it is necessary for some.special reason, the use of the most rapid plate or film (supersensitized) and the least possihl ' projection distance will give satisfactory results. The photographing of colloidal solids under the slit ultramicroscope can be accomplished without difficulty, as the exposure can be pro longed to the. desired extent. F or this reason, slower and more '('011 trasty plates can be employed. Another type of illumination which can be classed with dark (il'hl, because it is based on the same principle, is that known as Rhcinbcr ' illumination. This form of lighting ~,mploys two differently colored filters to produce the desired effeet. One of these (possibly of a blue calor) is a disk just large enough to fill the full aperture of the objective, This is set in the center of another calor (red, for example) of a contrasting nature, the outside disk being the full size of the substage conFIG. 13 I. Rhein denser. Such a composite disk is illustrated in Figbcrg Disk urc I 3 r, When such a disk is placed under the substage (the diaphragm being wide open), the object appears bril lianrly illuminated, the calor of the outer ring, while the field has Ihe color of the central disk. There is not much advantage in photographing in black and white with these disks, although if a considerable series of them is availah.lt, in various color combinations, some pleasing effects can be secured J ()J' -crruin objects. In deciding what combination is to be used, one IIIIISI merely consider that two filters ar being employed, and judge i hc cf f 'Cl Oil Ihe basis of what each filler will a -complish alone, when uSl'd wirh Iht' particular plaIt' ht'illg.l'llIployt'd. The id ':11 IIS~'of Rheil.' h 'I' r ilhuuinarion is 10 ht, fOllllll ill l'ollllt'l'lion with photo trap" III 1\IIIIII'all'olol'S, which \\" shall di '\I 11\1 'I.

208

SPECIAL PHOTOMICROGRAPHIC

PROCESSES

PHOTOMICROGRAPHY

WITH

POLARIZED LIGHT

PHOTOMICROGRAPHY

WITH

POLARIZED

LIGHT

C?ccasions arise in many types of microscopical work where illumination by means of polarized light is required. Chemical crystals, petr?graphic ~ections, starches, fibers, and various animal andvegeta~)Ictissues are included among the types.of objects which can be studicd to advantage by means of polarized light. All of these? at times, must be photographed as they appear under this type of illumination. 1\I~hoLlgh instan~es occur when surfaces of opaque objects must be examined, and possibly photographed, by means of polarized light, by far t he greater portion of work with this form of illumination comes in t he transl11itte~-light .class. It should, therefore, be treated just like :11l'yother transmitted-lighr work so far as photographic problems are concerned. The use of critical lighting is desirable for the best results, and t he effects of magnification, numerical aperture, and other factors' are identical. . 'I here ar.e, howe'.'er, problems in photomicrography with polarized Itg~1tpe~uhar to this type. of work. These relate to the presence of :lstJgmatlsm when a pnsm IS used as the analyzer, covering a large area 111 low-power work; correct rendition of interference colors in black and white; presence of extinction in some portions of a section when the polarizers are crossed; photography of interference figures; and others of like nature. When a modern high-quality petrographic stand is employed, some of these problems are automatically solved. For instance, astigmatism, c<IlIs.edby the presence o.f the non-symmetrical prism in the path of the image-forming rays, IS compensated by means of a cylindrically rround lens associated with the prism. Astigmatism is not so visually noriccahlc since one is subconsciously manipulating the fine adjustment and to some extent compensating for it. The effect of astigmatism is to make lines in the object which lie in one direction, out of focus when those at right angles are sharp. Altering the focus reverses the condition. 111 a photomicrograph when astigmatism is present, either set of lines can be sharpened, at the expense of the other, but a compromise foc~s is SOIIl<.'liIllCS pr Icrablc. I I)\It, to the high .osr of large prisms, and the scarcity of suitable iC9land spar with which to make them, it is cxrrcm Iy difficult to secure prisllls sldlit'it'lltiy I:trg' t() .ov 'I' low-power (ields and the full <lp'r t urc of micr photographic lenses, This problem has b 'en solv d for

many classes of work, in recent years, with the development of Polaroid and other polarizing materials of a similar nature. Polaroid can be substituted for both the polarizer and analyzer. Astigmatism is not present when it is used, but other conditions are introduced-which must be reckoned.with. Polaroid 'acts by the partial absorption of the rays vibrating in one direction. When the films are crossed, i.e., in what would be the 'position of .extinction, there is considerable residual light in the redremaining (and occasionally in the blue), and hence the effect is not one of complete blackness, but a decided red or purple-red color persists. Figure 132 shows the spectro-photometric graph of Polaroid, double in parallel position, 10111d cr.ossed. Except for slight surface reflection and transmission losses, prisms ofany type made from calcite will show a substantially straight line. 50 per cent loss for oneprism an.d zero transmission when cross.ed, These differences must be. taken into account whenphotographing with Polaroid: ' . . Nicol prisms of the conventional type with slanting faces are quite unsatisfactory as analyzers, for 100 photomicrographic work. The flat face modified form of Nicol 90 - P ldro 'd prism, Ahrens or Abbe prisms, L,...- v .are far superior. The Abbe 80 L> ~v. triple-prism type does not bend 70 J-.- !-the rejected ray at a very great. j angle, but it is usually suffi- i;; 60 [.4 l-. Fi ims parallel ciently far removed so as not to': I strike the plate.. ~ 5.0 1/ One of the most serious corn- .~ I plications encountered In the ~ 40 ~ photographing of rock sections h 30 I1 is the difficulty of eliminating , the condition of a: considerable 20 , 1 percentage of minerals lying in I1 the approximate position of ex10 Filrns ~d ,Y. tinction. No matter how a seco ~~~~~~~~~~~~~~~~ tion may be turned, as one grain Wavelength in Millimicrons becomes Ijght, another darkens. f ibl I' FrG 132 Spectra-photometric Curve of Ir is 0 ten POSSI e to arnc rotate . . Polaroid Films this situation either by turning
I CrOSS

210

SPECIAL PHOTOMICROGRAPHIC

PROCESSES

PHOTOMICROGRAPHY

IN NARROW SPECTRAL REGIONS

2I I

the aI?alyzer slightly out of the 90 position, or by inserting a selenite plate m the system, between the polarizers. . Panc~1romatic plates. should be used for polarized-light work, but even with .these .there IS !l0 assurance that a proper differentiation of rh? ~olors .111 the Image WIll be obtained in the picture. The reason for this IS ObVIOUS.Two mineral grains may.lie side by side; one a bright g~'een and the ~)ther a rich red, visually, in the-position chosen for the pi 'Hire. Yet If the total.transmission of light in each happens to be of Ihe same order, they will photograph alike and not even the bound:\1' .1> n~een t?em may show. An eye trained to appreciate variations 111 Itght ,111tens~ty, apart from color, is a great asset in this work, for it llSll.ally ISpossible to find a rotational position where maximum differellt~atlOn on the basis of i?tens~ty will prov~de the best pictu~e. I hotography of polarized-light effects m natural color IS the ideal solution, wherever possible. _' Photograp~ing int~rference figures in crystals is simple when a microscope equipped with a Bertrand lens is available. Where an ordinary n:icr~scope is employed and the analyzer is located directly above the objective, ~ B~rtrand lens can often. be improvised by mounting a low-power objective (about 2" focus) m the threads at the bottom of ~I~c .dra~v-.tube. The latter is the.n used to o.btain the correct focus by slIdl~g It m and out. Conoscopic observation of interference figures ~'eqlll~es~he use of at least a 4 mm. (40X) objective and a full cone of d!11111I11a~lOn ~rom the condenser .. Any arrangement which will pro~'Ille a visual interference ~g~ue will suffice for photographing it, but 11ll!l1Sr. he understood that It ISthe Bertrand lens and not the objective which ISto be focussed to obtain a sharp image.
PIIOTOI\IICROGRAPHY IN ARROW AND MONOCHROMATIC SPECTRAL

REGIONS

. Ph{)togr~phy in narrow spectral regions is but a specialized modificatl.()~l of ordinary ~olor filter work. It differs, so far as the region of the visible spectrum ISconcerned, largely in the nature of the lizht sources employed. ' b .As a matter of fact, only two of t~lese arc of interest to the photolI11c~ogr:'I~h.er.Both arc, to.all practical purposes, strictly monochro1I1:ltIC. I his mono .hromnri . charara 't ristic is quire different from till' 1I:1ITOW('SI band possible with filters, since but one spe ific wave I'll rth is utilized.

F or one of these an electric sodium lamp is employed. In a lamp of this type (Figure 52) a little sodium metal is vaporized by means of a heating element until the electric current will arc across two terminals through the sodium vappr, which is made to emit its typical spectrum. The spectrum of sodium consists of very few lines in the visible region, all of which are of relatively small intensity, except that known as the D line, -which is an intense yellow. * When a sodium lamp is used as the source of illumination no filter is required, since only light of substantially a single wave length is emitted. Photography by means of the yellow D line is of value under two different conditions. One is that chromatically, at least, the performance of the objective IS perfect, even though the lens itself is far from ideal in its correction for chromatic aberration. There can be no chromatic aberration when there is but one wave length of light involved. If, therefore, the spherical correction in this region is good, a fine image results: But a still more important advantage may lie in the relative absorption by different elements in an object, of a specific wave length; hence a radical differentiation may be achieved in a photograph, such as is scarcely, or not at all, evident to the eye, and which will vanish completely, ever;t on a photographic image, when a slightly wider-spectral band is employed. This stage of photomicrographic research is still in its infancy and may result in many new discoveries when it is more fully employed. (See Plate 29 in Chapter 8 as an example of this.) The other wave length in the visible spectrum which is available and frequently employed is a powerful green line present in a mercury arc lamp. The mercury lamp is somewhat similar in principle to the electric sodium lamp, but no preliminary heating is required, since mercury is a liquid at ordinary temperatures and can establish its own circuit to form the required arc. There is one essential difference between the mercury and the sodium lamps in their application to photomicrography .. While mercury only emits a few wave lengths in the visible region, there is not the same difference in their relative intensity and they are more widely scattered. Moreover, there is a very powerful line in the ultra-violet which has more effect on a photographic * The D line is one of the series of prominent black (absorption) lines seen in the solar spectrull1. Actually it is double, but the two lines are so close together (5889.97 and 5895.93 ngstrorn Units) they usually appear as one in small spectroscopes. Two Of her pairs of lines are also present, one on each side of the D line (5683-5688, and ()I ~4 01(1) but they are not sufficiently strong materially to affect the plate during the I imc required 10 mal c :1Il cxposur' with the I) line.

, Il

SPECIAl.

1'1I0'1'0 M ICROGRAPHIC

PROCESSES

PHOTOMICROGRAPHY

IN

NARROW

SPECTRAL

REGIONS

21

emulsion than any other. For this reason a filter must be used in combinurion with a mcrcury lamp to eliminate all but the desired line. The i'.:lstlll:m \N rattcn filter # 77 cuts out practically all but the green line at H()o a.u. This line is passed at about 70 per cent .efficiency, so that \IT have available another monochromatic light source, in addition to t he Sodium 0 line. II would be equally possible to isolate the 4358 a.u. (violet) line in I Ill' Illt"l"t"llryarc, by means-of a suitable filter, but the relative insensi11\ II\' (If I h ' cye to the blue-violet region makes focussing difficult, or 1\ I1 impossible. Since this is the case, there is little advantage in using 1111 11I1l',because, by an ingenious method, which will be discussed III I, II i: pt)ssiblc to utilize the strong line lying completely within the lilt I I ICdl'lIT rion, at 3650-3655 a.u. (double). Either panchromatic III IIIIItul'ltrolll:tric plates sensitive to green and yellow must be em1"11\ d wi: It hoth the sodium and mercury (gre~n) lines. It i c arc I wo narrow band regions where photomicrography can h' 'alril'd 1111 10 advantage with ordinary equipment. These are the 11 -ur 11 It rn violet and the infra-red. BUIIt \\' rarrcn and J ena glass filters are available for these regions. For the ultra-violet end, though mercury vapor lamps can be used, arc laIII ps will provide a more intense source; if direct current is available, the most powerful source of all lies in the iron arc, operated at around five alllpcres. Ordinary glass lenses will transmit light between 3000 and 4000 a.u. ' although the balsam used for cementing the components will fluoresce :t lit! le. Microscope objectives are not so well corrected in this region nnd it can bc expected that the focus will not be the same as for the shortest blue-violet rays which the eye can use (which, of course, are loturcr t hun +()()O a.u.): One cannot be sure, until a test is made, just how rood all image can be produced with his particular lenses, but it is wor: h Irying, when .vcr an occasion arises where a picture in the ultra violet IT rion is desirable. Then' an' Iwo possihlc methods of determining the focus. The 1'1\ f '1.11>k,.uul l>y far rhc quicl er one, 'when the illumination is suffiI .ntlv inrcns, is hv mcuus of a strongly fluorescing glass plate of III 111111111 'LI~s. ()IIl: of Ihe hcs: for th ' purpose is the jcna glass filter I . \ oIa~suf Ihis I)'pl', IIIOUl1ledon Ih ' plan ' of rh s .nsitizcd (I, '. ill lieu of t ItI' } round glass, which IlIllSI he removed) and d h\ 1111',1110, uf fo('ussill} rlnss (Figure 7(,), will r .v 'al the imI 11 fhllll " -iuu Oil I It, ..]:ISS. \ Vhcrc this rauuot he done il is

necessary to make a set of test exposures, preferably by means of a multiplicator back, to determine the proper position, The procedure is as follows: ./ < First, secure the best approximation of the focus by means of the blue-violet (C) filter; replace the latter with the ultra-violet filter and run a series of test exposures to determine the proper exposure time. Having found the latter, note the position of the graduations on thefine adjustment knob, rotate it a few microns so as to back the objective away from, the object, andrecord the position. A series of test strip e~posures should now be taken ona multiplicator, starting with the recorded position of the graduations on the fine adjustment, the knob being turned so as to bring the objective one micron toward the object in each subsequent strip. The total number of strips should be such that the position of best .visible focus with the blue-violet filter will lie somewhere near the central exposure. This strip test will indicate in which direction from the visible position, if there' be a divergence, the correct ultra-violet focus lies. It may, by chance, show the proper focus. Should one of the end exposures appear the best of' the series, a second test plate should be made to extend the exposures past the correct focus. One may also judge, by the difference between the individual exp9sures of the first test, whether a larger spacing or a still finer step should be used. In general, low-power objectives are not so sensitive to slight variations in focus, while high-power oilimmersion lenses require intervals of the order of one-half micron. When once the variation between the visual blue-violet and ultraviolet focus has been determined for a given lens, it should be recorded in the 'photomicrographic notebook. Subsequent micrographs can then be taken by the simple expedient of focussing with the C filter, moving the graduations on the fine adjustment knob in the proper direction the amount of correction necessary; and then changing filters and exposing. When Zeiss apochromatic objectives are available it is not necessary to follow this procedure, since this firm supplies auxiliary sleeve lenses to slip inside of the regular series to correct them for use in the ultraviolet region. They also make a similar correcting lens for use in the inf ra-rcd. lrra-violct work does not require color-corrected plates; the slower, fincr-grain plates of the process and near-process class are supcrior and equally fast in the hort-wave region. It is not to be exPl'l't .d Ihat Ihe type of ultra-violet work described will be used

214

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES

PHOTOMICROGRAPHY

IN NARROW SPECTRAL REGIONS

21

especially for increasing resolution, since the cl;aracteristics of the objectives, other than the chromatic focus, are not altered by the correction in focal position. The special advantage in this type of. ul.traviolet work lies in the demonstration of, and utilization of, vanations in the absorption of ultra-violet light in the region of 3000 to 400.0 a.u. This, of course, is band absorption, as distinguished from absorption at one definite wave length. . Just as it is possible to work in the short-wave region lying beyond the visible range, so can we proceed with the non-visible rays at the opposite end, the infra-red rays.. Infra-red filters covering different bands in the infra-red reglOn are available in both the W ratten and J ena glass series; these can therefore be chosen soas to utilize the particular region ~esir~cl.. ~lre p~ocedl~re as to establishing the correct focus for the obJectlv.e IS Ident1c~l ~lth that described for ultra-violet photography except 111 the substituting of the F filter for the C in the visual pari: of the operation. . .. An arc lamp should not be used for infra-red work, S111ceIt IS relatively poor in the longer waves as compared to a tungsten lamp. To obtain an equivalent effect, there is much excess vi~ible and ultraviolet illumination to absorb, A tungsten lamp, working at reduced voltage, is superior. It is desirable to use a light ~hich pr.ovide.s enough visible red to accomplish the focussing; otherwise a strictly infra-red glow lamp would be ideal. , Infra-red photography requires photographic plate~ corrected for use in this region. Such plates must be orde~ed speclal~y, fl:om the Ial't urv , as they are not a stock item. Tho;.e interested 111 this work 1ICIllld secure from the Eastman Kodak Company a copy of their pub1I1'at ion, " Photographic Plates for use in ,Spectroscopy and AStr01~0111\ ." Figllre T33 is a chart of plate sensitivities taken from this hool let, For most photomicrographic work in the infra-red, the type. 1'1 plate shown on this chart is ideal, and is within the range here ordinary apochromatic objectives may be expected to funcIion sat isfactorily. . Far more rnicroscopical research work with both the ultra-VIOlet and infra-red bands should be done than has been attempted to date. Much data of a scientific nature remains to be established by these aids. Another type of work closely associated with ultra-violet light is fluorescence (or luminescence) microscopy. The invisible shor waves of the ultra-violet region arc able to s r lip svmpnrh .ric vihrarions of longer wave lengths when they fall on certain substances.

'When these longer wave lengths are within the vibration range of the visible spectrum, they become evident to the eye and the substance emitting them appears luminous. This phenomenon is known-as fluorescence when the visible. light effect ceases quickly after the exciting ra s are cut off, and as phosphorescence when the reaction per'sists for an appreciable time longer.
tASTMAN
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TOTAL S'ENSITIVITY SPECTRAL REGION

ESPECIALLY

FIG.

133

Only a limited number of substances fluoresce to a degree sufficient to make them of interest from the microscopical point of view. Minerals and chemicals furnish the most brilliant examples, but instances arc not lacking in the animal and vegetable kingdoms. Fluorescence ofT crs additional means of identifying certain elements, tissues, etc., and also another method of differentiating various micro-structures. In re .cnt years, considerable progress has been made in fluorescence 'lIi('J"oSC01)Y through the treatment of tissues with fluorescing chemicals for whic 1 there is a sclc .tivc effect in various structures. Ithough

an

216

SPECIAL PHOTOMTCROGRAPHIC PROCESSES'

MONOCHROMATIC

ULTRA-VIOLET

PHOTO 'lICROGRAPHY

217

no change may be apparent to the eye, the effect under ultra-violet light is analogous to differential staining in ordinary microscopical preparations. Depending upon the type of work being done, fluorescence microscopy may, or may not, require considerable special apparatus over that needed for ordinary work. In the first place, some form of lamp rich in ultra-violet rays is essential. The preferred sources are either an arc lamp (preferably with iron electrodes) 01' a quartz mercury vapor lamp, or both, since the effects are somewhat different with each. There are now on the market special ultra-violet bulb lamps which suffice for visual work but do not possess sufficient intensity for photomicrographic work. . Fluorescence microscopy can be divided for consideration into two groups - that with incident light, and that where transmitted light must be used. The systems of illumination do not, in theory, differ from those where visible light is employed. The ultra-violet is secured by the use of either Wratten or Jena glass ultra-violet filters. As most optical glass transmits considerable ultra-violet, the glass lenses (condensers, etc.) can be employed with fair results. Where maximum illumination is desired, however, replacing all the glass in the illuminating system with similar elements of quartz materially increases the brilliance of the fluorescent effect, since the absorption of ultra-violet through several thick glass lenses is con~dcrabk. . For visual work, no further change in the illuminating system is necessary. With only ultra-violet light striking an object, in both incident and transmitted setups, the field should appear dark when no fluorescing object is in place, but wherever fluorescence occurs, the effect should at once be evident. But photographs cannot be taken under these conditions, since ultra-violet light, to which the eye is insensitive, can be both transmitted and reflected by the object, and if allowed to reach the plate would register an image of its own. It is imperative, therefore, that every bit of ultra-violet light be prevented from acting on the plate. This is accomplished by the use of a filter placed over the front of the objective or somewhere in the system, between the object and the plate. Unmounted sheet gelatin filters are often advantageous for this purpose, as their thickness does not interfere with the performance of the lenses. The exact lilt r to be chosen depends somewhat on the ultra-violet filter employed. It is essential that there be no overlapping of the two. suully the

Wratten #8 (K2) suffices. For strictly transmitted-light work, cover glasses are available which act as filters to remove the ultra-violet, but when these are used, there is no opportunity to examine the object by means 0 incident light projected onto the top surface. A supersensitive panchromatic plate or film should be used forthe photography of fluorescent effects so as to obtain full rendition of the light emitted, which frequently contains considerable red.. Also, the total light available is very feeble and a long exposure is necessary even with fast plates. For practically all types of fluorescent work only low powers can be used, as otherwise exposure times may extend into many hours. Because of this condition, photomicrographic work in the field of fluorescence microscopy has been, up to the present, of extremely limited application. 1\110
'OCHROMATIC ULTRA-VIOLET PHOTOMICROGRAPHY

Photomicrography with monochromatic ultra-violet light is radically different from that previously described, where a fairly wide band is employed. In monochromatic work, since a single wave length is utilized, special optics can be designed to give ideal correction at the specific. wave length, without regard to meeting conditions throughout a wide spectral region. The primary purpose in working with monochromatic ultra-violet light is to secure greater resolution, for, as pointed out in Chapter I, the shorter the wave length of the light employed, the larger the number of lines per inch which can be resolved . . Two practical methods have been developed to utilize a monochromatic ultra-violet light source. The simpler, and at the same time, less expensive, is that of Bausch & Lamb, which utilizes the 36503655 a.u. lines in the mercury vapor arc. The only additional equipment necessary is a quartz mercury vapor lamp and a set of special objectives. These special objectives, instead of being corrected over a wide band, are designed to be chromatically and spherically corrected at two wave lengths only - the 3650 a.u. ultra-violet line and the 5460 a.u. green line of the mercury vapor lamp. To meet such correction is relatively easy, whereas, if a lens were required to function equally well at all points between these limits, compromises in the computation of the lens would have to be made 'which would lower its p .rfonuan .c ~H the desired ultra-violet \V,1\Ielength. The great 'Si single ohst acl to over '()I Ill' in the t cchniquc of photograph

218

SPECIAL PHOTOMICROGRAPHIC

PROCESSES'

MO TOCHROMATIC ULTRA-VIOLET

PHOTOMICROGRAPHY

21

outside the visual range is the difficulty of focussing the object on the plane of the sensitized plate. With the Bausch & Lomb special lenses, the focussing is accomplished by the aid of the green (5460) line and a filter which passes only this line. When the green image is in focus, the ultra-violet image formed by the ultra-violet (3650) line is likewise in focus, so that changing filters is the only additional operation necessary in order to make an exposure with ultra-violet. The green and ultra-violet filters are furnished as part of the complete Bausch & Lomb ultra-violet photomicrographic outfit, which is illustrated in Figure 134. The immersion fluid employed with the high-power oil-immersion objective is not cedar oil (as standard for visual work) but sandalwood oil. When the highest possible performance with the oil-immersion lens is desired, it is a good idea to make a check on the exact focal point of the ultra-violet as compared with the green. This check is made by the method described for ascertaining the divergence of the ultraviolet focus when working with ordinary objectives, outside of the visible range. The divergence, if any, will be found to be slight, and a factor only when photographing such objects as the markings on amphipleura pellucida, or a ruled grating, for maximum resolution .. Because of the likely small divergence, the steps in the test exposures should be of the order of t to t micron, and it is not necessary to extend the test beyond two microns on each side of the green focus. Comparative tests on a given specimen (e.g., of amphipleura pellucida) reveal at once the great increase in resolution obtainable with the short wave length. * The transmission of very thin films of the majority of mountants for the 3650 a.u. line is such that most objects can be photographed, regardless of the nature of the mounting medium. But there are some limitations. For instance, realgar, the best medium for finely marked diatoms from the standpoint of refractive index differentiation, possesses such strong absorption in the ultra-violet region that it is ruled out for all ultra-violet work. The nearest high index mountant which
* It is not feasible to make direct comparisons, even of the same species of diatom (amphipleura) when photomicrographs have been taken from different slides, because of the wide divergence among the individual diatoms. This is especially true when they are not from the same locality. Even in a single slide of specimens from the same locality, there will be found some difference in the dcgrcc of sharpness of the . markings between various individuals whcn oriented in thc same position. Id .al specimens showing the dot structure sharply defined arc rare.

220

SPECIAL PHOTOMICROGRAPH,IC

PROCESSES

MONOCHROMATIC

ULTRA-VIOLET

PHOTOMICROGRAPHY

221

can be used is hyrax. This absorbs considerable ultra-violet, but can be used if a sufficiently long exposure is given. The. alter?ate method of photographing with a single line in the ultra-violet IS one developed many years ago by the Zeiss Company, although without its ever experiencing an extended 'demand. The reasons for t~is are several. In the first place, it is primarily a research tool, intended largely for one specific purpose - acquiring further l~nowledge of m~cro-structures through increased resolution. It does gIve remarkable Improvement in resolution under the limited conditions where it can be used, but there is no record of a sinzle new fact, brough~ to light throu~h the superior resolution it provide~. The eqUIpment IS very expenSIve, as compared with any other microscopical apparatus on the market; its cost can therefore be justified only i.n occasi~nal instances, since its use is very limited. Further, the technique of ItS operatIon calls for knowledge and experience far beyond that possess~d ?Y: th~ rank .and file of microscopical workers. Reg~rdless o~ all this, It IS still entitled to rank as one of the highest of microscopical developments, and other uses for it have materialized, which entirely eclipse the original idea of increased resolving power,

2796 - 2798 - 2803 a.u., in the magnesium spectrum. Since there are so many of these powerful lines together, the illumination is much more intense, although theoretically the performance of the objectives is not of so high an order, the light not being strictly monochromatic. The relation of the cadium and magnesium lines is shown in the spectrogram of these metals (Figure 135)' :
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In t.heory. the apparatus is quite simple. By the use of a high-voltage el~ctncal discharge across e~ectrodes made from a metal possessing WIdely separated but strongly spectral lines, in the ultra-violet, a ~uitable .source o~ light is obtained. The.metal chosen for the purpose IS cadmium, which has several strong lines, the most suitable being. that at 2,749 a.u. This is far below the transmission limit of ordinary and optical glass, such as that employed for microscopical purposes, and hence is useless with regular microscopical lenses. Moreover, it cannot be passed through an ultra-violet filter of the Wratten or Jena types, such as those used with the Bausch & Lomb outfit. Recourse ?lust ~e had to c~ysta~ quartz for every portion of the optical train, including the collimating condenser, microscope substage condenser, all objectives and eyepieces, and a quartz prism substituted for the ~irr.or. In addition, the isolation of the 275 mu line (as the 2749 a.u. lIne. ISusually called) must be accomplished by means of quartz prisms which separate the lines sufficiently far that only the 275 line can enter the quartz substage condenser of the microscope. The microscope objectives are highly corrected for the 275 line, but as long as the light is monochromatic, other lines can also be used. The most common substitute source is a group of four lines ar 279 I -

FIG. 135. SPECTROGRAM OF CADMIUM A D MAGNESIUM Showing the spectral lines of magnesium (top), cadmium (middle), and iron (bot tom) in the region between 2200 and 4500 a.u. The single cadmium line at 2750 and the several magnesium lines at 2800 are those chosen for photographing in the ultra violet with the Zeiss outfit.

As no visible light is present, preliminary location of the desired field is secured by the use of an auxiliary light reflected into the substage from the face of one of the monochromating prisms. For rough . focussing with the ultra-violet light a special viewing device termed a finder is placed over the eyepiece. This contains a fluorescent screen and a magnifier for viewing the image formed on the screen. When uitably adjusted the image is in focus at a fixed projection distance when the fluorescent image is sharply in focus in the finder. Best results are obtained when the power source is alternating cur'rent, using a stepup transformer K.V.A.) operating at 10,000 to 15,000 volts, in combination with a tuned circuit. Where only dire .t current is available an electrolytic or mercury interrupter must h . provided, in combination with a high potential spark coil. The latest form of the outfit, with fixed electrodes, is shown in Figu re I 30. Since its introduction the apparatus has undergone continued hangt (If mechanical design, although the optics remain substantially un changed.

et

222

SPECIAL PHOTOMICROGRAPHIC

PROCESSES , Mono '-PICTURE PHOTOMICROGRAPHY 223

di . 1. . g con mons w rich circumscribe the f I' ;~f~r~~~eaf:;s~~c~~ted with the poor transmission of most ~~~s~antc~: '. . g .275 ft Glass, all common mounrin . d' 1 maJonty of tissues, all stains, most inorganic chemical com~:::r~;:,' a~J

Many of the limitin

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Zeiss Ultra-Violet

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substances in which the microscor " in Ihis ultra-violet region Hence ~dlS.t ISlilte~ested, appear opaque , . no 01 lilary rrncrosco 1 IIOIl cnn . pIca prepara. be studied by this appa1atus. ObJccts IllUSt be mounted on quartz slid' . colorlcss petroleum oil (Nu' 01) or I . es, ltl a medlU~ such as cover glasses TIll J ~ ycerme, and covered With quartz ",. le srna quartz slides f I' I . ". mcral carrier in Figur b ,one 0 w lIC 1 IS shown on Its covers arc ' e ) 37, .cost a out $2'50 each and the Y' .ir .ulnr IlIlies,'in' higl~~:~Wt~nC~~~:~, h~tv~Sr~l~~~l;I~'\~~i~li~l~'~vt~~~II'~~(r :r ~~l:l.t. ~l~I.)~;i: c le:lpt'!' and s:ltlsf:lclOry for 1ll:1llyIYl1 S of '1 '1'1' . se .I~C. :11 . WOIc. icrr t r:II1SllllSSIOII

01 her

is around 75 per cent of quartz, for the 275 mu line, but falls off rapidly below this. From a practical standpoint, the. greatest value of this ultra-violet equipment is in the differentiation of various micro-constituents through the degree of absorption present. For instance, if one had a mixture of powdered glass 'and amorphous (fused) silica, the proportion of each could be quickly determined: the glass would photoFIG. 137. Rock Crystal Object Slide, graph black, while the fused quartz with Metal Carrier particles would be transparent. It is possible by the use of this apparatus to differentiate between the cytoplasm and nucleus of unstained, living cells, since the nucleus strongly absorbs ultra-violet. This opens up a means of studying morphological changes taking place in the living cell, and the transitional steps between hving and dead protoplasm. Although the entire operating technique of this apparatus calls for the consideration of many details which cannot be discussed in our limited space, most of the general processes are similar to those already considered. As. with other cases where the image cannot be directly observed on the ground glass, tests for critical focus have to be made. Tests to determine the correct exposure obviously must be made at the beginning of the work, after which computed exposures can be relied upon for variations in magnification. Computed exposures, however, are of no value when one must differentiate between two constituents through their absorption characteristics, if the percentage of transmission in both is of a low order. Plate XL VI in Chapter 8 is an example of this sort. Very slow, fine grain, non-color-corrected plates, such as the Hammer Slow, or Process, plates are best suited for work with ultra-violet light.

MOTION-PICTURE

PHOTOMICROGRAPHY

Though the field of motion-picture photomicrography is considcrably restricted, it is of extreme importance for revealing what takes place under certain conditions. Original applications of motion piet urcs through the microscope were confined largely to displaying livin r or mnisms - protozoa, etc. - in water. Such pictures were wel-

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES

MOTION - PICTURE

PHOTOMICROGRAPHY

come additions to various educational films, in the standard 35 mm. class. Recent motion-picture work has been largely confined to the J6 mm. class .and mi~roscope manufacturers have developed apparatus nnd .accessones for .It. The smalle~ film pos~esses several.advantages ~)VC[ the st.ar:~ard SIze,.for photomicrogrnphiwork. Chief of these IS the possibility of USl11ga less intense light for the same field area

/1'/(1.

138.

Bausch and Lamb Outfit for Motion Picture

Photomicrography

(()vl -r cd,

since tl:e li&"hton the film is inversely proportional

to the

;":l':t of the tW? SIzes, 1.e., about fiv.e times as great on the I6 mm. film. I h~ls for ,cql1l".alent exposure a light of one-fifth the intensity will
Sit(.1H.e: 1<,xce~sIve concentration of .light, with its attendant heat, is ~)bJe~tronable ill the case of many rnmute organisms and delicate livIng tlSSlICS. Death frequentlyensues very quickly under intense light :In." heat ~I:d the reactions w~Ich may l~ave been photographed under this condition cann?t be considered typical. . Among the specIfic classes of work especially adapted to motionplCtl1J~C photo.m.Icrog~aphy can ?C mcntion~d (in addition to the photorraphll1g of hVlng micro-organisms). StudICSof blood circulation and r,he flt.llcriol1.of .the various blood cells in rebllilding rissu " eel] proIifcrnrion (miroric processes, crc.), crystal rrowrh, chcmicul rcuct iOlls,

Brownian movement - in fact, anything where changes in structure or relationships can be shown to occur over a period of time. The camera required for motion-picture photomicrography must be a standard commercial model from which the lens can be removed. It is not possible, as in other types of photomicrographic work, to connect the microscope directly to the camera, since it is necessary to have the field being photographed under observation at all times. The_ intermediate fitting, known as an observation eyepiece, is therefore an important and essential portion of the equipment. For this reason, it is a better policy to purchase the outfit as supplied by the various' manufacturers than to attempt to construct it of nondescript parts. The Bausch & Lomb outfit is illustrated in Figure 138. It is designed to operate with the Eastman Cine Kodak. (vario~ls models). ~ny standard microscope can be used, but special eyepIeces are required. When a rapidly moving object that is apt to leave the field of vision is being photographed, it is essential that the microscope be equipped with a mechanical stage. It is frequently necessary to be constantly manipulating 'both the fine-focus adjustment and the mechanical stage ) while such objects are being taken. . 'Some of the rapidly moving organisms may be photographed with " slow' motion." (i.e., 64 frames per second) with advantage, but the light intensity should be increased accordingly. It is possible to slow down the motion of organisms considerably by adding to the water a viscous medium, such as gelatin, egg albumen, solution of slippery elm bark, gum arabic, etc. When the problem concerns the photographing of change~ taking place so slowly that no movement can be observed over a consldera?le period of tim,e, and a period of m~ny h~urs, or even days, may be I.nvolved in the total cycle to be depicted, It becomes necessary to deVIse special apparatus for the purpose, or secure one of the elaborate ou~fits which have been made available by some manufacturers. What IS required is a clock-driven timing device whicl~ will tU~'n on the light and make a single-frame exposure at predetermined periods. In other words, instead of making exposures at the rate of I6 per second, they may be one per second, one per minute, or even one per hour in extreme cases. It is the opposite of slow motion, since when the frames are shown at the normal rate, what occurred in many hours may be shown in a minute, or less. Knowing the time interval between exposures, one 'can compute the elapsed time in any particular cycle. by .ounring the number of fram s from start to finish.

226

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES

PHOTOMICROGRAPHY

COLOR

227

, Details of setting up and operating may vary considerably, dependmg upon the make of equipment secured, and hence recourse must be had to the instruction booklet issued by the firm manufacturing the ?utfit., ,Th~ fun,da~entals of general photomicrography as to securmg critical illumination, use of filters, determining exposure times, etc., apply equally well to motion-picture work.

PHOTOMICROGRAPHY

IN

COLOR

Any color process used in ordinary photography is equally adaptable ,to phot0l!llcrography, Some, however, are subject to limitations w~lch make It more difficult to secure correct color rendition, though this ma~ not be S? serious a condition as when portraying natural colors, since the pnmary purpose of color in microscopic work is differentiation of structure. . From the standpoint of final results color processes can be divided mt~ two classes, t!lose producing a colored print on paper and those whl~h must be viewed as transparencies. The former require the making of color separation negatives, while in general the latter is accomplished by means of a single exposure. The following are the more commonly used processes. (1)

scopic filter of its parti,cul~r colo:', for tha~ portion of tl~e image fall~ng upon it. The entire Image IS a mosaic of these minute filtered Images. After development of the exposed film, it, must, be reversed ,by the method of dissolving out all the darkened silver ~mage, exposmg ~he undissolved silver to a bright light and redeveloping. The result IS a positive image, which, when viewed by, transmitted light p~s~ing through the mosaic colored screen, appea:'s m the colors of d~~ original. Special compensation filters are f~ul11s~ed, by the Lumiere Company, for use with different types of illumination. When th~s,e filters are properly matched to the light being used, the color renditions are quite accurate. , ' Since the starch grains of the color screen ab~orb considerable light, exposures are from 30 to 60 times those .reql~ued for, ord~nary l2anchromatic plates, without filters, and a fairly intense light IS required for autochromes when they are projected as lantern slides.

(2)

The Agta Color Process

The Lumiere Autocbrome

Originally the Lumie~e Autochrome was supplied as a plate but it has been changed over 111 recent years to a celluloid film base, The color is produce? by the ~lse of a screen of dyed starch grains applied ~o the film base, m a varmsh layer, over which the sensitized emulsion ISpla~ed. The starch grains are dyed in three colors, blue, green, and r,ed, 111, the proper pr?port,ions to yield an approximation of white hght (~.e" a neutral nnt, S111ce the absorption is considerable). As the grams average around. lOI 5 mm, in diameter, there are somewhere ' around 3,000,000 of them in one square inch, The interstices between the grains are filled with carbon black, for which is claimed an effect similar to the addition of a fourth (black) impression when electro~~~~or. ' , Exposure of the film is made through the back of the film, since all light must p,a~sthrough ,the layer o,f colored starch grains before srrikmg the sensitized emulsion. In this way each grain becomes a micro"

The Agfa color process is similar t? th~ Lumiere. The screen? instead of being composed of starch grams, ISmade up of dye? pa,melcs of gum arabic, fused into the varnish ,laye~ so, that each pamcl~ ISsu bstantially circular. The ~verage pa:-t1C,le,Slze ISa?Ol~t the same 111 both, but the Agfa screen particles vary individually 111 SIZeto a far gre~tcJ' degree than those of the Lumiere. Figure I39 shows a con:pans~)I1 , of the two screens, at a magnification of 300X. The gum arabic grall:s are farmore transparent than the starch graiI1:s,and no carbon black ~s used with them; hence the Agfa film reqUlres less exposure and IS considerably more transparent in the final positive. Unfortunately for the photomicrographer, the color balance in the Agfa plate is unsuited for many types of W?:-k. The ~reen prc'dominates, and though this is a favorable condition for Or?111ary outdoor photography, it makes a poor ~ack~round f?r photo,m~crographs. The place where Agfa plates excel m lTIlCrOSCOplC work IS 111 polarizatiun effects. The compensation filters for Agfa films, are radically difFerel,lt f~()m those used with Lurnicrc films, for any gIVen source of illumination, and hence cannot be used interchangeably.

228

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES

PHOTOMICROGRAPHY

IN

COLOR

229

(3) The Dufay Color Process


This i~ another of the color processes of the same general type as the Lumiere and Agfa. The color screen in this case is a reseau of fine

inch, the graininess of the mosaic background is much coarser than in the Lurniere and Agfa, so that the transparencies will not stand so much enlargement as lantern slides. The transparency is of the same order as that of the Agfa film. The general technique is the same in all three and they all have the advantage that they can be handled in all stages of the process by the photomicrographer himself, and individual pictures can be taken, developed, and studied before proceeding to take the next picture. The total elapsed time from the taking of the picture to the binding up of the finished transparency need not exceed an hour or so. .

(4) The Finlay Process


The Finlay process is a revamped Paget process, which was on the market for many years. The essential difference lies in improvements in the color correction of the screen and minor improvements in the technique. As in the Dufay process, the screen is a printed one, but the squares are 'much larger and very little enlargement can be employed without the screen becoming evident. The squares run a little over 300 to the inch or about 100,000 to the square inch. The relative appearance of the Dufay and Paget (Finlay) screens is shown in Figure 140 at the same magnification (300x) employed for Figure 139. The Finlay process is quite different from those previously described in that the col or screen is not an integral part of each plate or film, but is a separate unit, known as the taking screen." The plate can be an ordinary panchromatic plate and the cost of the negative is therefore not greater than in ordinary black and white photography. For taking the negative, the taking screen is placed directly in from of the plate, screen side next to the emulsion, and they are held tightly in contact in the plate holder. The negative is developed in the regular manner and from it glass transparency positives are printed by contact. A viewing screen" is required for every positive. These are similar to the taking screen except that they are not so free fr0111 printing defects, and hence are considerably cheaper. The viewing screen must be carefully aligned with the positive so that the colon; rebister exactly as those of the taking screen register with the ncgarive. A fter registry, the transparency and viewing screen must be bound together so that subsequent shifting cannot occur. \Vith this pro .css, ';IS l11any dupli ar rr:lIlsparcn .ics may be made as r 'quir xl, whereas with the pro .csscs ill which the .olor S(.'J' .cn is

FIG. 139.

Pr'IOTOMICROGRAPHSOF LUMJERE AND AGFA COLOR SCREENS

The calor screen of dyed starch grains of the Lurniere Autochrorne plate is shown o?- the left and the screen of the Agfa plate on the right. Both are magnified 300 diameters.

rul~d lines and squares, in the three colors. While the ruled lines, which are produced by a printing process, run about rooo to the lineal inch, or the equivalent of 1,000,000 squares of color to the squar'

230

SPECIAL PHOTOMICROGRAPHIC

PROCESSES

EASTMAN

KODACHROME

231

integral with the plate, each picture is a unit and cannot be duplicated except by rephotographing on a new plate. In any of these color proce~ses which the photomicrographer may elect to tryout, the first step should be the making o~ test exposures for

formation booklets issued by the manufacturer covering the specific process, be carefully studied and followed, unless one has had previous experience in the working of the process in other lines of photography.

Safety Film

Support Antihetetion ~"'-----~~-Backing FIG. [41. Cl'OSSSection of Kodachrome Film

F[G. [40. CaLoR SCREENS OF THE DUFAY ANDFINLAYPLATES The color screen of the Dufay film is shown on the left, and that of the Finlny 7111 Pngcr) plate on the right. Both are at a magnification of joox, hence arc dir cily comparable with the Lurniere and Agfa screens shown in Fig. J 39.

determining the proper exposure time. As the re omrncnd d d vel opers, developing technique, and other it ms in the pr 'para! ion of rhc transparency vary somewhat in each case, ir is important that the ill

equal for serious work of the larger sizes in the Professional series, Type B (the artificial-light film). Unlike the other processes described, the color in Kodachrome is not produced by mosaic patterns, and the only graininess present is that of the silver emulsion. The structure of the film is shown in cross section in Figure 141. The most serious drawback in the use of Kodachrome lies in the fact that the films must be sent to the factory at Rochester, . Y., for processing and unless one is wining to pay a considerable premium for having them processed singly, they must be sent in lots of at least three films at a time. This means not only a considerable delay, but 11 degree of photomicrographic experience adequate to assure freedom from failures in exposure. On the other hand, the actual work involved is less than with other col or processes. Since the type B film is adjusted for working without a filter, with nrtilicial-light sources having an intensity rating of poaK, it is imwr! nnr that the proper lamp be chosen as the source of illumination. li rh-inr nsity gas-filled tungsten lamps come within the allowable limits. The 50o-watt size is ideal. Photoflood lamps can also be used. '

SPECIAL PHOTOMICROGRAPHIC

PROCESSES'

SEPARATION NEGATIVE PROCESSES

Some variation in the color of the finished Kodachromes can be expected in various lots sent to be processed. This is apparently due to the impossibility of controlling the numerous steps in the toning processes. The predominant color may run to either the blue, red, or yellow. It is not objectionable for photomicrographic work, and is conspicuous only when pictures in a single series but processed in different lots, which one would expect to be similar; are compared . side by side. Col or work with Kodachrome, in photomicrography, especially in the large sizes of film, off~rs a beautiful illu~tr~tion of the. valu.e 9f computed exposures. In spIte of the great vanation of magl1lficatlons, which may extend from two or three diameters up to several thousand, and the wide divergence in time, which may be I/2sth of a second or several minutes, there must be substantially no failures.: One can allot a few films to experimental purposes, when starting work with ~odachr?I.ne, but when once basi.c exposure times under widely varymg conditions, have been determined, from then on it should be clear sailing. Though there. are other color processes of the 'single print transparency class, chiefly of the two-calor type, these are hardly of interest to the photomicrographer. Digression from those described will more likely be into the field of three-color separation negatives. Separation Negative Processes

. The basic principle invol,:,ed in processes where separation negatives are employed, ISthe taking of three negatives of the same object, through blue, green, and red filters, from 'which transparencies can be made and toned in the complementary colors. These three transparenc~es when superimposed yield a reproduction of the object, in approximately natural colors. The transparencies can be arranzed to provide a finished transrarency (i.e., for use as a lantern slide) o~ they can be mounted on white paper as a colored paper print. As tJ'iany duplicates as desired can be produced by these processes. For ordinary commercial photography various methods of obtaining the separation negatives have been devised. These are largely a~ong lines.th~t provi?e for the simultaneous exposure of all three negatives. ThIS IS essential because of the probability of movement occurring between exposures if they are made one at a time .. Usually such conditions do not exist in photomicrography, and

hence it is possible to expose three separate negatives, using respectively the A, B, and C filters out of the standard Wratten M set. The negatives must be made on panchromatic plates or films. The exposure times for each negative will vary, depending upon the exact plate and light source employed. With Eastman Panchromatic plates and a soo-watt high-efficiency tungsten lamp, the exposure ratios will differ for each batch of plates, but may be in the order of 10 for the red, 25 for the green, and 50 for the blue. The red and green should be developed for identical times, the blue somewhat longer. Several different techniques have been developed for producing the final picture from the separation negatives. Photographers who have had previous experience in this type of work can easily apply any one desired to photomicrographic work. Those taking up such work for the first time will find the simplest approach to success via the Defender Chrornotone process. F or this process a stripping paper has been developed by the Defender Photo Supply Company, of Rochester, N. Y. Black and white prints are made on this paper in the regular manner from all three negatives. After development and fixation of the prints, continued soaking causes the collodion film (which is quite similar in superficial appearar).ce to cellophane) to separate from the paper support. The paper is thrown away and the three prints on the collodion are processed so that the silver images are removed and replaced by dyed (or more properly, toned) images. The print from the negative made 'with the red filter is toned blue (blue-green), that from the green negative is toned red (magenta) and that from the blue negative, yellow. These three colored films are superim.posed upon each other on a specially coated paper and, when dry, produce the finished colored print. Complete details of the process are available from the manufacturers. When one has mastered the principle involved in separation negative color work, through the Chromotone process, other methods, which are standard and described in many works on photography, can be tried out if desired, although better results than those possible with the Chrornotone process can hardly be expected. All color-separation work in photomicrography is limited by a scriOl'S condition which is easily overcome in ordinary photography, but for which there exists no remedy when photographing through the microscope, This relates to the density to which each of the prints I11l1stbe printed and developed so rhc .ornbination of the three will he properly balanced when slIlH;rllllpos xl, In ordinary photography

234

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES.

STEREOSCOPIC

PHOTOMICROGRAPHY

235

this is taken care of by means of a suitable color chart and neutral tint scale which can be included at the edge of the picture (although trimmed out of the finished print). This serves as a reference and the density can be determined accurately by means, of it. 0 such device can. be included in a photomicrograph and there is no way of telling, until the three prints are finally superimposed, whether they are correctly balanced. As a matter of fact, they seldom are, but if the colors are brilliant, it often does not matter if the final print does not correctly interpret the original. The most serious condition exists when the background, which ought to be colorless, or yellowish, shows a decided red or blue. This can be anticipated, to some extentrby the amount of color in the background of the red and blue prints, which should be negligible.
STEREOSCOPIC PHOTOMICROGRAPHY

of various planes in it, accurate in all things except the exact relation of height to breadth, a lack of orthostereoscopic reproduction is often not a handicap. This is fortunate, since in some kinds of work a magnification of many hundred diameters is required, yet a stereoscopic image of some sort is essential, even if it be not possible to produce a truly orthostereoscopic condition.

Instances constantly occur in microscopy where it is a difficult and at times well-nigh impossible task to interpret properly a structure depicted in a single photograph. The value of stereoscopic pictures which can be examined in an ordinary stereoscope, in such cases, cannot be overstated. Several devices are on the market for taking stereoscopic photomicrographs, but they are largely designed for low-power work. Camera attachments are available for mounting on Greenough binocular microscopes, or carrying the paired Greenough objectives. The Bausch & Lomb Co. have developed the Ortho-Stereo camera shown in Figure 142, which, with a complete set of lenses, takes pictures from full size up to 24 diameters. This apparatus, in combination with the special stereoscope designed for use with it, gives a true threedimensional picture - that is, the object appears to the eye just as it would if uniformly increased in size, the same amount as the magnification. Unless attention is paid to this particular feature, the magnification of the third dimension - i.e., the depth - may not actually coincide with the lateral enlargement. If the vertical dimension enlargement is less than the lateral, the height of the object will appear less, in proportion, while if the vertical enlargement is greater than it should be, the object will appear higher in proportion to its size. Since in both these conditions, however, a three-dimensional, plastic image is produced which gives a proper conception of an bject and the relation

As a rule the two pictures comprising a photomicro-stereograph cannot be taken simultaneously, although, if some specific problem warranted the expense involved in constructing special apparatus, it could be accomplished by the utilization of one of the methods hy which stereoscopic pictures can be produced. The only apparent need for such a device would be the photographing of living or ~n()v ing matter. Very low-power stereos can, of course, be taker: sJl1~lIlrancously with the Greenough binocular, but where the objectives alone are employed, and no eyepieces are present, the results are not ery satisfactory. On the other hand, since there is little need for pictures of living organisms, and since nOl~-motile subjects c~n be photographed in two separate exposures, this latter procedure IS en"

rircl y snrisfa wry. . Stereoscopic or binocular

VISUlIl,poss .sscd by all individuals With

..

..

SPECIAL PHOTOMICROGRAPHIC

PROCESSES

THE

ELECTRONIC MICROSCOPE

237

normal eyesight, results from the superimposing of two different views of an object, into one composite picture, by the co-ordinating power of the brain. When an object is located at the distance of best vision from the eye (i.e., IO"), the parallax angle between the images is about 15 the exact angle depending upon the interpupillary distance of each individual. The requirement for a stereoscopic picture is that two views be taken from points separated approximately the distance between the ~yes with th~ obje~t seemingly located ten inches away. For an obJect at full size this means that the angle between the two views is ar?und 15 If. the .angle be materially less than this, the apparent h~Ight of, the object ISlowered although a three-dimensional effect is s~Ill o~tamed. C?n.versely, a g:reater al:gle accen.tuates the height. SI~lce,.m gene~al, ~t IS not essential, especially for higher-power work with smgle objectives, that true orthostereoscopic effects be obtained, any one.of several,different methods ca? be employed with an ordinary photomicrographic outfit, to secure satisfactory results. . . There is considerable separation in the angular view of an object picked up ~y t1:e semicircular areas. of an objective on opposite sides of the median line, so that by the simple expedient of covering onehalf of the back lens of the objective to take one view, and the other half to take the second picture fairly good stereographs can be produced. A similar result is obtained by covering one-half the image at the Ramsden circle. This is the method utilized in the Abbe stereoscopic binocular eyepiece, as made by Zeiss. Better results are obtained by a displacement of the image on the ground glass an amount equal to the interpupillary distance (i.e., about 2fr"). This method requires a camera large enough to take the two VIews on one plate. It also requires the use of a mask which will cover one-half the. plate. while the corresponding half is being exposed. The other pIcture IS then taken by reversing the mask, making sure, of course, that the change in the position of the object has also been made. ~he shifting 0' the i~age can be accomplished either by means of a shift of the object WIth the mechanical stage, or if a centerinz objectiv~ holder is available, the objective can be displaced late~ally, each SIde of the center, the proper amount. It is preferable to locate th~ c~nter of t~e desired area in the center of the ground glass, then shift It to the nght (or left) an amount equal to one-half the desired
0 , 0

displacement for the first view, and in the opposite. direction an equivalent amount beyond the center, for the second pIcture. By far the best results are obtained when .a tilting stage can be ~sed. Such an item is not available as standard eqUlpment at the present time, but possibly will be in the near future. It can be ma.de by anyone handy in the working of metals. A sketch of the one designed and used by the author is sho:vn in F~gur.e 143. The secr~t of .a successful design is to have the lme of plvotmg cross the OptiC aXISof the ml.croscope and at the .s~me time lie exactly on the plan~ ?f the object. When these conditions are met, there IS no lateral shifting of the obSpring

----,.

Cl; ps

Pivoted at level of object:

./

....Base to be secured to Stage

FIG. 143.

Tilting Stage for Stereo-Micrographs

ject. When the stage is level, the view is not ~ifferent from that with the object lying on the reg:ll~ st~g.e of the ml~r?SCope. !o t~ke th~ stereo pictures, the stage IS.mclmed ab?ut ~ I~ one direction fo! the first picture an~ then 7 1l~ the OpP?SIte direction, for the second. o mask is used, since both views fall m the center of the plate, but smaller plates can be used. . , In microscopic work, as in ordinary stere~scopIC photography, t1:e prints must be reversed in mounting, otherwise a pseudo-stereoscoplc effect results. Both plates should have the same expos~re and should . he developed together so as to yield prints of equal density.
0

THE

ELECTRONIC

MICROSCOPE

The most recent development in microscopical .and. photomicrographic apparatus and one that appea:s d~stIned to YIeld l?lpo~tant re .sulrs along certain lines is the electromc microscope. ~hlle still SO~l)~what in the experimental stage, it has reached the pomt wh~re It IS being put on. the market in a commercial form by the Radio or1,,)(,:11 ion of merica.. .' . .. The underlying principle m the elect:-omc microscope ISthe utiliza I ion of .xrrcmcly short 'wave lengths, l.~". of t1:e order of !/! OO,()()O of t he I 'ngrh of rh shortest wave of visible lrght. hcs a re e\cc

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES

THE ELECTRONIC

MICROSCOPE

239

visible light. The image is seen visually on a fluorescent screen, and can be photographed on a plate. Practical magnifications up to at least 25;000 to 30,000 diameters yield good resolution. Pictures have been taken much higher than this but with considerable "empty
11:16._

r,ll"

8 .. AI ,.,,;,.

FIG. 145.

Photomicrograph (B. Anthracis-Original Magnification . One-half) Taken with Electronic Microscope

35,000.

Reduced

FIG. 144.

R.e.A. Electronic

Microscope.

volragcs such a~ th~sc employed in

typc produced by vcry high -ray work. Th re arc no I 'I1S s III r.he el.ectrol~lc microscope, their pia .c bcing talc n by clcctroumg lien' 'oils whi .h can ',HIS' the paths of the cl '('I rons 10 follow d '(I nire cours .s, jllst :IS a glass lens ':111 cont rol ihc direct ion of u bc.uu of

tronic discharges of the cathode-ray

magnification," since the same laws operate with the electronic microscope as with ordinary microscopes. The limiting factor, at present, in the electronic microscope, as regards the ultimate magnification, lies in the effective numerical apertu re of the system, which cannot approach that of a high-apertured oil-immersion glass system. If it could, magnifications up to 400,000x or 50o,ooox would be possible, without appreciable empty magnifiC:lt

ion,

I .ikc rh
depend

ultra-violet microscope, the electronic instrument must .xclusivcly Oil rh absorption differential of the object for the

240

SPECIAL

PHOTOMICROGRAPHIC

PROCESSES

p~rticular .frequency empl~yed.. For this reason its use ~s extremely circumscribed. Many of its pictures are shadowgraphs, or silhouettes. On the other hand, objects like unstained bacteria, cells, etc., ~hich cannot be seen to advantage with ordinary light, yield fine Images. The electronic microscope will probably always remain a research tool of limited application, not only because of the relatively few uses for it, but because of high initial cost (at present around $9,000), high .ost of oper~tion, and the involved t.echnique of operation. The entire electronic flow must take place m vacuo, so that even the object, which has to be specially mounted on a gossamer-like film of nitro.cllulose, must be in the vacuum. Variations in magnification are achieved by changing the strength of tbe magnetic lenses. Figure 144 shows a view of the complete instrument, as standardized by the Radio Corporation of America. Figure 145 shows a photomicrograph taken with the electronic microscope.

Chapter 6 MICROPHOTOGRAPHY The term microphotograph is frequently employed, even by some microscopists, as the equivalent of photomicrograph. This is incorrect, as the word means" a minute photograph." .. Photographs reduced to microscopic size, capable of being seen 111 detail only under a microscope, are not novelties: for th~y have lo~g been in practical use. They were of great value 111 the Siege of Pans, during the Franco-Prussian War of 1871, when, by the employment of the Pigeon Post film, messages were passed back and forth between the besieged city and the outside world. The f!1essages were fir~t printed in newspaper form and then reduced to 1l;1l1utephotographic copies on celluloid film. Th~se fil~s were rolled 111tO small tubes and attached to the leg of a homing pigeon. . When received they were read by means of a microscope. Although these hastily executed miniature ,Publications cannot ,rank as works of art, it is remarkable, considenng that photographic technique of seventy years ago was not so nearly perfect~d as it is today, how legible the copies were. Figure 146 shows a ITIlcrog,raph .made from an authentic piece of this Pigeon Post film, at a magnification of 50 diameters. ' Many improvements in microphotograph~c technique have been introduced since the days of the' Franco-Prussian War and some wonderful commercial work has been done. Until recent years, however, most of it has been confined to the production of microscopical sli~es of riiicrophotographs. These illustrate to the amate~r mlCr?SCOplSt the art of microphotography; examples can be found 111 tl~e slide cO.lle .rions of the majority of the older gene~ation. A. ph~tomlcrogr~phlc copy of one of these microphotographs ISshown 111 FIgure. 147. Th.e original measures .77 mm. by I.l.mm. (less than 1/16" hIgh) and it is magriificd 85 diameters in t?e mlcrograp~1. , . They have also been exploited as novelties, 111 the form of a minute hollow-rub' holder in which is placed a micrograph mounted on the 241

MICROPHOTOGRAPHY

MICROPHOTOGRAPHY

243

end of a glass r.od wit~ a strongly magnifying lens at the 'opposite end. T~e enlarged Im.age IS seen by looking into the eye end of the tube, ~'1th the tub.e pOl1~ted toward a bright light. From the strictly scientific standpoint, microphotography has been of value in the production

practice. Reduction of such printed material to minute dimensions by means of microphotography has been found to be a practical solution. By this means a ten thousand volume library can be reduced to the size of a few ordinary books, and a complete newspaper is as easily stored as a postage stamp.

FIG. 147. Copy of a Microphotograph" FIG. 146. Photomicrograph of Pigeon Post Film, Paris 1871, 50X

Zephyr and Flora," 85x

of .minutc instrument scales, stage micrometers, eyepiece disks of va no us typcs, etc. I n rc~cnt years. there has been a gradual appl ication of the Fundamcnt al Id .a of mIcrophotography to library and .ornmcr .ial proh It'IIlS. Mainr .nan ., of a cornpl I filc of records, n 'wspapcrs, bool s, und other dO(,1I111 .ius has gro.wn 1'0 such r rcmendous proport ions Ihal SIOl':l 't' rOWll C:ll1l1ol ht provided for un uuliruiu] ('0111 inuuncc of Iht,

The microscope can lay claim to microphotography as its legitimate offspring, because some form of microscope is essential for the examination of a microphotograph after it has been produced. For the examination of microphotographic copies of books~ pap~rs, und the like, numerous viewing devices have been and are still bemg developed. The more elaborate of these restore the copy to the full size of rhc original and ar fully auromari . in operation, to the extent ,'hat :111 entire hook l11:1y lie rend throu rh, just as easily as though it

MICROPHOTOGRAPHY

THE LENS

w~re a full-size library edition. An entire page is projected on a suitable viewing screen so that it is all visible at once. Elaborate means have also been developed for the commercial production of microphotographic reproductions of publications, on a large scale. The amateur microscopisr, however, interested solely in the process as a means of making microphotographs on standard I" x 3" microscope slides, needs very little apparatus beyond his standard microscope equipment. All this can be - or rather, must be - homemade, as there is no standard outfit on the market for this purpose. Thus very little expense is involved. The making of microphotographs is an interesting side line for the photographically minded amateur microscopist. Probably much more would have been done before now, but unfortunately very little has been published about methods and there appears to be a common fear that the process is too complicated to be undertaken by the unskilled. Actually it is extremely simple and can be mastered by anyone sufficiently interested to take the steps involved in setting up for the work. A brief description of methods is included here so that all phases of photographic work in which the microscope plays a part can be said to be covered. In principle, the making of microphotographs does not differ from ~he making of lantern slides from original negatives of larger size. It IS essentially the degree of reduction that is involved, but because the re?uction is so extreme in amount, and the finished transparency so minute, three new factors are involved in the production of first-class pIctures. These are: (I) the quality of the lens which must be employed in the reduction of the negative; (2) the need for accurate focussing, and (3) the use of a transparency plate which has a substantially grainless emulsion when properly developed.'
(I)

Tbe Lens

It is obvious that the limit of 250 microns on the size of the circle of con[usion, ordinarily considered satisfactory for photographic purposes, ISabsolutely impossible in microphotographic work. When the reproductions are unusually small, a magnification of 100 diameters may be required to show them properly. If the pictures are to be clear and sharply defined at this magnification, the 250 micron limit cannot be greatly exceeded in the magnified image. This means that rh circle of confusion in the microphotograph itself cannot exceed more than a few microns in diameter.

Fortunately the microscopisr usually has, in his standard equipment, lenses which should meet this requirement. These are his low-power objectives, and microphotographic lenses. The latter are, for reasons which will be evident later, to be preferred where they are available. Naturally the smallest microphotographs can be produced with the shortest-focus lens, other conditions being equal. As explained in Cha]?ter I, unsym~etrical lenses must be e!11pl?yed in the way in which they are designed to operate. Hence It ISImportant, whatever lens is used, to have the conjugate foci of the lens which has been designed as the shorter, pointed toward the image, when the latter is a reduced image, as is the case when making a microphotograph. With low-power microscope lenses, and microphotographic lenses (microtessars, etc.), this means that the front of the lens, 'which is toward the object in taking a photomicrograph, must be faced toward the plate, in making a microphotograph. Should one contemplate buying a lens especially for microphotographic work, the best is a high-quality motion-picture lens such as those used on 8 mm. cameras. It need not necessarily be one with a high aperture; preference should be given to that for which the smal~est circle of confusion is claimed by the manufacturer. A lens of this type will be faced toward the negative which is to be reproduced, and the plate will occupy the position of the motion-picture film. Ordinary microscope objectives up to 16 mm. (IOX) can be used but ge'nerally the image will not be ideal. The reason is that such lenses are corrected for use with a cover glass and for a definite tube length. To obtain the best result the negative should be located at the distance of the eyepiece diaphragm from the back of the lens, and the picture should be taken with a cover glass in front of the sensitized film, which would occupy the position of the object, in relation to the objective. Omission of. the cover glass adds to the tube length (i.e., the position where the negative should be situated), but the latter would still be too close for practical work, because theoretically it would require a negative only ten to twelve times the size of the micrograph to be made from it. With a lens not exceeding IOX, however, considerable leeway will exist, even when ideal conditions are not pr .scnt, so that fairly good pictures can often be obtained by obscrvIIlg the conditions outlined later on. Objectives corrected for a tube k'llgth of infinity will be found :0 wOI~kmuch bett~r. M~ny such :1I'l' 1l0W provid .d for use on sp .cinl <:QUlp1l1 nts. With ordinary objectivl's:I partial correction cun hl' drl'Ctl'd h the use of a ne rnrivc spn't:H'k klls of one or two dioptl'l's, loc;ttl'd jllst ill front of the oh

MICROPHOTOGRAPHY

SUITABLE PLATES FOR lVJICROPHOTOGRAPHS

247

jective (i.e., actually at the back of the lens, as it would be in the-microscope which becomes the front in taking micrographs). A negative lens extends the tube length, giving an approximation of the desired longer conjugate focus. Blank (circular cut) spectacle lenses usually can be secured from an optician or eye specialist. Such a lens must, of .cour~e, be a simple negative lens, without any correction for astigmatism. (2)

The Need for Accurate Focussing

The fact that the microphotograph is, in effect, a microscopic object which must be examined at considerable magnification, is sufficient explanation why a perfect focus must be secured before the exposure is made. A safe rule to follow is that the image of the negative should be observed while being focussed on the plane of the film, at the magnification which will be used later on to show the finished micrograph. For instance, if the size of the image is such that it just nicely fills the field of the microscope at lOO diameters, it should be examined and focussed at this magnification. Logically this implies that the entire process be carried out under a microscope. This is the crux of the entire process. The practical working out of the method by which it is accomplished will be outlined further on.

(3) Suitable Plates for Micropbotograpbs


The third imperative condition to be met in the production of microphotographs is the practical elimination of grain in the plate. Ordinary plates are not suitable, for the sensitive silver emulsion, instead of being homogeneous (or rather, so finely emulsified as to appear homogeneous, at fairly high magnification), is very grainy, the silver being present in the finished negative in minute blackened particles which produce the image. Hence the image, although satisfactory at normal size, or slightly enlarged, is hardly recognizable when highly magnified. A general law relating to graininess in ordinary photography is that fast (or very sensitive) emulsions are inherently coarser than slow ones. This implies that the very slow process, or lantern-slide positive, plates can be expected to represent the finest possible grain to be found in commercial plates, which is true. Much has recently been done, largely because of the popularizing of miniature cameras and the introduction of reversible 16 mm. motion picture film, ill the

reduction.of grain size in fast and supersensitive emulsions. Reduction of grain size has not been carried out to the same extent on very slow emulsions, largely because there is no need for finer grain in this range in ordinary photographic work. The work on emulsion grain size has been supplemented by the introduction of fine-grain developers. Where necessary, the use of fine-grain developers on slow emulsions gives as grainless a transparency or positive reproduction as can be desired for all usual purposes. Such fine grain is, however, not suited for microphotographs. With ordinary process plate emulsions, even if the graininess could be completely eliminated through proper development, other conditions are present' which make them unsuited for microphotographic work. These are the thickness of the emulsion, and its turbidity. These two factors, operating together, result in a spreading of the light, so that, microscopically speaking, a diffused image results. . Fortunately, for the microscopist desirous of experimenting with microphotographic work, there is available a commercial plate which, for all practical purposes, is suitable, providing magnifications not in excess of 100 diameters are used for viewing the microphotographs. This is the" Alpha" Lantern Slide plate, manufactured by Ilford, Ltd., London. The emulsion on these plates is extremely slow, very thin, and almost transparent. They should be developed in the exact developer recommended by the manufacturer. These plates are stocked by many of the larger photographic supply houses in this country. . The only commercial plate of American manufacture, of which I am aware, which comes into the grainless class of the Alpha plate is the Eastman Spectrographic plate, Type V. These plates must be ordered specially from the factory. They are, like the Alpha plates, extremely slow, and, as they are intended primarily for spectographic work, are extremely contrasty. By overexposure and soft development they will suffice for microphotographs where contrast is not objectionable, and are especially fine for ruled scales. The ideal emulsion for this type of work is one of the old-style al\H1I11en emulsion, or next best, the collodio-bromide type of plate, for these are substantially grainless. As these are not made commercially, the entire pro .css of making the emulsions, 'oaring the plates, and ,r 'p,lring rh 'Ill for use must he P .rIorm .d :IS a part of (he work. This umdi 'ap IS likely \0 d.uupcn the urdor of i hc most enthusiastic mi-

MICROPHOTOGRAPHY

THE METHOD

OF

'lAKING

MICROPHOTOGRAPHS

249

croscopist. If, after experimenting with Alpha plates, one enjoys the hobby sufficiently to go the limit, he can then take up the coating of his own plates. Formulae for such emulsions can be found in many works on photography. The Method ot Making Microphotographs Only two pieces of equipment are essential to the process. These are the microscope and the illuminating device for the negative. The latter can be designed along anyone of several lines, but must be, to some extent at least, homemade. From the standpoint of ultimate effect, it can be described as a light source, in front of which is placed the negative, the whole suitably enclosed so that no light, other than that passing through the negative, is visible. Figure 148 shows three possible arrangements, all equally satisfactory. From the operating point of view, they differ only in the relative intensity of the light available, which affects the time of exposure. The exact time with any of these systems can be varied over a considerable range by merely changing the power of lamp employed, and hence that design can be chosen which will be the easiest for one to construct with the facilities at his command. At A is shown the conventional condenser method, as used for stereopticon projection. This provides the highest light efficiency, but if the condensers must be purchased especially for the purpose, it is the most expensive of the three designs. The design shown in B is next in cf- ficiency as to light intensity, ind is satisfactory when the negative is nor large. A large negative may require more than one lamp to provide uni- _ form illumination, or the single lamp must be placed at a zrcarcr distance FIG. 148. Designs of Lamp fr0111 the opal glass. nl 'ss a great dis House and egative Carrier for Taking Microphorogrnphs ranee e:111 exist het we .n t he lens and

negative there is not much chance of a large negative'S being satisfa torily copied. The efficiency of this design is increased by painting the entire inside of the housing with flat white paint. The indirect illumination used in the design shown in C provides an ideally uniform lighting of the negative, but the efficiency is low and longer exposures are required. Whichever design is used should provide for easy' insertion and removal of the negative and should have the light shield in front of the negative extended sufficiently far to reduce the stray light entering the room. Either wood or sheet metal can be employed to make the housing. . Where possible, a uniform negative size should be established, since this simplifies the design of the apparatus and the entire setup need

of Photo.::.ra~~h=1_================J ~ from Focal of


Sizo
'>I.

Size

of

Negative

Lens

lenath:

~DU~'~ta~n~ce~0~if~N~eg~a~tiv=e Lens

FIG. 149.

Relation of the Objective Focal Length and Negative Distance, to the Si".' of egative and the Resultant Microphotograph Size

never be. changed, except for a possible variation in the distance of the negative from the lens, when larger or smaller pictures are desired. When a miniature camera of the Leica type is available, this size of negative will be found ideal as a standard size for all work. The actual . holder for the' negative can be made to accommodate a larger size than this -- possibly up to the size of a o. 120 (Brownie Kodak) film. The wisdom of exceeding this latter size, however, is questionable, for usually it will be found that the practical distance which can be provided between the negative and lens limits the negative size materially. Before deciding upon the size to be used, it is well to take .stock of all factors having a bearing on it. Figure 149 illustrates diagrammatically the relationships involved. Working out a practical example according to this diagram, if we assumc a lens of one-inch focal length to be used, and a picture one millimeter square to be desired, a negative d" square would need to be placed three feet from the lens, to secure the proper result. \ Vhar iv r size of negativc is used, it must he so framed in thc holder that 110 .xt mucous light can P:ISS it. The framing ;l 'wally forms t he
lillliting borders of the niicrophoto rJ':lplt. !thollgh tht' IIli('l'oscllpt' is :lIll'ss'l\tial p:lrt of

the cquipmcnr,

tht'

MICROPHOTOGRAPHY

THE

METHOD

OF MAKING

MICROPHOTOGRAPHS

pict~res are .not.made througl~the mi~roscope, which is employed primanly for vlewmg and focussmg the Image. The lens used for makinz the pictures is to be mounted in the substage condenser ring, in plac~ of tl~eregular c?ndenser. Usually some form of adapter must be made sp.ecIally for tl115purpose, but it need not be expensive. Where Zeiss ITIlCrOSCopes are available, the centering objective holder for the substage, which is standard equipment with this company, can be used fo: all lenses equipped with the Royal Society screw thread. Lenses WIth other threads must be fitted with adapters to fit the objective holder. !hose who hav~ mastered the principle of critical illumination 'will ql11ckly g~'asp d~e Idea of t~l~entire process. The microscope must be placed m Its honzontal posltlon and the illumination box with the negPlate (em"lsion next) to stage)~ ,.. Negatwe Lens mounted / in substage Filter Lens focussed. by substage 11wve'ment Baseboard FIG. 150.

._____7

(when required)

Lamq House

Diagram of Complete Setup for Taking Microphorographs

ative i~lposition, emulsion sid~ out, must be located in the optic axis of the 111lCrOSeope,. the proper distance away, to yield the desired image. Though a special base or support for the two pieces of apparatus is r:ot ~ssential, it simplifies matters appreciably, if much work along this line IS contemplated, to provide a heavy board with guide strips and cl.amps, for this purpose. The setup is shown diagrammatically in FIgure ISO. TI:e microscope stage is the support for the plate on which the picture IS to be taken. The emulsion side is placed directly against the. surface of the stage .. This means that the focussing of the microscope on the plane of the Image must be done through the thickness of the glass plate. As any variation in this thickness affects the focus/it is important that the focussing be done in every instance on the exact plate on which the microphotograph is to be made, but usually one focussing opera~ion will suffice for all exposures made on the same plate. The partlcula~ method of focussing to be adopted will depend upon whether an entire plate 3" x I" is used for each picture, or s veral are taken on a strip which are subsequently cut lip to be mounted

on ordinary glass slides. The latter is by far the less expensive, but does not make as neat a finished slide, and involves more work in finishing the slide. If the plates are purchased in the standard 3t" x 4" lantern-slide size, they must be cut to the proper size by means of a glass cutter (preferably a diamond) as the first operation. This must be done in the darkroom, but a fairly bright orange light can be employed. The Eastman #0 safelight is suitable. If 3" x I" slips are to be used, the first cut should be a t" strip off the 3ttl dimension, to reduce the plate to 3" x 4". This t" strip should be saved for focussing purposes. It can also be used for- making test exposures. The 3 x 4" glass is then cut into four 3" x I" strips; these are packed away in a box until they are required, to prevent fogging. With this method each originallantern slide will produce four finished slides. ' If the alternate method of making separate small pictures is to be used, the lantern-slide plate can be cut into six strips, along the fourinch length. These strips should not be cut into the smaller squares until after the pictures are finished. Each strip will make at least four, which will give twenty-four possible pictures to a plate. Having provided plates cut to the de~ired size and aligned the ~pparatus, we are now ready to proceed WIth the actual work of making the exposure. . The room in which the work is being done must be capable of bell1p . darkened to exclude all light that would fog the plate, as the latter IS not mounted in a holder of any sort, but placed directly on the stage of the microscope, film side down. A ruby or orange bulb in one of the light fixtures will provide ample but safe illum~nation, as the plates are very insensitive to any but the blue rays. (ThIS does not apply, of course, to the Eastman spectrograph plates, Type V.) In placing the plate in position on the stage care should be taken, not to slide it on the emulsion, as scratching of the film may result. Fach time it is moved, it should first be raised from the stage. With rh ' . 3'" x I" slide plates, focussing can be done on either e?-d, or. anywhere except within a t" circle in the center, as the emulsion Will later be removed from the slide in all but the central area. With a plate in position on the sta.ge, the first op~rat~ol1 .isthe focussing of the microscope on t.he emuls.lOn. A. IOX ob)ectlve.Js preferable for this purpose. If any ~lfFiCLlltyIS expenenc~d 1Il findl.ng the 'XH'! focus, a minute scratch With a nccdl WIll provide an object. \Vh .n Ihe ~"S! rip is sa cd it should Ill' used for! his purpose. her the micro
11

MICROPHOTOGRAPHY

THE METHOD

OF MAKING

MICROPHOTOGRAPHS

253

scope has been foc~ssed ?n t~e film the focussing adjustments must not be touch~d aga~n whIle. slId.es.fron: the same plate are being exposed. In dOll1g this focussing It IS desirable that the lens in the subs~age condenser, with which the picture is to be taken, be thrown entirely out o~ fo~us so that there is no temptation to focus on the image of the negatIve instead of on the emulsion surface. In other words we n:ust bril~g the image of the negative to the proper focus 0~1the e~ulsI~n (which means bring it. to the point of sharpest definition in the m~croscope) aft~r the latter ISpr?perly focussed. This is accomplisbed with the focussing means provided for the subsraze condenser. Instruments provided only with slip-ring condensers will naturally be more difficult to adjust to proper focus. In the diagram (Figure 150) a filter holder is indicated located directly in front of the ne.gative. If the correction of the p110tographic lens IS not ~o perfect as It should be, the blue rays, which are the only ones affectll1~ t~e plate, may n~t possess the same focus as the optical center of white light. When this ISthe case the introduction of a blue filter (of optical quality) will aid in obtaining the proper focus for the blue rays. Th~ filter should then be left in for taking the picture. * Havll1~' obtall1ed. the proper focus of the negative on the plate, the next ?tep ISthe making of test exposures, which should be made in geometric progressIOn, I, 2,4,8, 16, etc. It may even be necessary to make a second set .of test exposures, and finally to change the lamp so that the prop.er time may fall somewhere between ten and sixty seconds. For making the test exposures it is permissible to slide the plate along, for on test plates scratches are immaterial. The test exposures can be spac~d about ~" apart,. in order .tha~ a considerable number may be po~sIble on a.smgle. stnp. Examination of the wet strip, after developmg and fixing, Will show the proper time. When 3" x I" slips are used, the picture should be located in the center. Small squa.res shou!d be so positioned on the strip that a single cut between each pIcture WIll re~ult 111 a neatly centered object. After the plates are dry, the pIctures should be covered with a cover glass and Canada b~lsam (in xylol), and set aside until thoroughly hard, The edges of the slide can then be ground smooth on a coarse India' or
" This applies to the" Alpha" plate and special albumen or collodion emulsions. If t~e ~astl~an .Spect~ographic plat~,. Ty~e V, is used, with the usual panchromatic ~ensltlzll1g, It WIll regIster al~ the VISIble lIght. everthcless an improvement in the irnage may result, ev~n 111 t~1JS case, through the use of a filter giving :l narrow band. It need not necessarily be 111 the blue, however, as a green band may be still IIIOI'C effective.

carborundum hone, using thin oil or turpentine. The final step is to scrape all the emulsion from the uncovered portion of the slide, and then clean and label. With small squares the additional work involved lies in cutting the individual pictures apart, grinding the four edges of each, and then cementing them to standard slips with balsam. The entire process can be a fascinating hobby and many pictures can be produced as a result of an evening's work. More seriously, the making of special scales for various scientific measuring instruments is frequently called for. Such scales or ruled designs must be accurately drawn in ink on white cardboard and then photographed on the proper-size negative. No attempt need be made to keep the scales to a definite size in the negative, as final adjustment in this case is to be made by altering the distance of the negative from the lens until the image measures the proper size. To effect the measurement of the scale on the microphotograph, an eyepiece micrometer must be employed in the microscope. This micrometer must be accurately calibrated beforehand, by means of a stage micrometer.

THE CHEMISTRY

OF DEVELOPMENT

255

Chapter 7 PHOTOGRAPHIC PROCESSES, MATERIALS, EQUIPME T A D

The Chemistry of Development The possibility of producing a permanent negative image through the use of various salts of silver is due to the fact that such salts are unstable under the action of light, They tend to break down into metallic silver, which, when dispersed in a solid emulsion, will then exist as microscopic opaque particles. When an intense light is allowed to act for a sufficient time on such an emulsion, the light alone is capable of producing reduction to metallic silver, but in some manner not yet fully understood, a faint light is also capable of initiating the process of reduction, the effect being prorortional to the intensity of the light. The most plausible explananons appear to be either that atomic nuclei of metallic silver are originated by the light (their number and size depending on the light intensity), these nuclei functioning as attraction centers upon which additional silver is deposited by reducing agents, or that some weakening of the bond between anions and cations occurs which aids subsequent reduction by chemical means. The particular silver salts of value in photography are those in which the metal is combined with one of the halogen group of elements, chlorine, bromine, and iodine. The incorporation of these silver compounds - silver chloride, silver bromide, and silver iodide - into the gelatin emulsion belongs to the plate- and film-maker's art; it is not necessary for the photographer to know the underlying chemistry, other th,an to know ,th~t plates and films can be produced which possess certarn characteristics, as to speed, softness, etc., in varying degrees. The one feature common to all, however, is their capacity to receive \~he latent image. This latent image, not visible in the undeveloped state, Can be acted upon by certain chemicals until it blackens. The degree of blackening is proportional to the amount of light which was allowed to fall upon the emulsion, The process of producing the blackened image is called developing,

Great divergence in the sorts of objects which must be photo~raphed so as to reveal their nature to the best advantage, calls for considerable latitude in the development and subsequent pri,nting processes, Or, to express it in photograph~c terms, so'n1~ negatl.ves must be developed for extreme contrast, while others reqUlre maximum softness. Althouzh the desired result can be obtained in part by proper selection of th~ plate or film ~mployed, in larg~ measure the d.evelopment can also be controlled to lmprove the quality o.f the negatlve, ,As development is a chemical process, an understanding of the chemistry of development helps to determine proce,dures, , The chemicals used in the production of the blackened image are employed in an aqueous solut~on,. technic~lly known. as the " developer." This developing SOIU.tlOl~ lS nO,t a smgle ch.enllca~, but.a composite mixture of several, which m vanous proportlons yield dlffe~ent results. A knowledge, therefore, of the part played by each constituent is useful to all who desire to become competent photographers" , The process of transforming the silver from the sal~ to the ~etall~c state is known chemically as reduction. Any chemical which will bring about this change is cal,led a .red:ucing agent. Therefore, ~ne constituent of every developing solution must be su.ch a reducing agent. Many of these are available; most ~re orga11l~ cou:pounds. But, for several reasons, none of these reducing agents 1Ssatisfactory for the development of an exposed plate, when us~d alone. .In the first place, reduction will ?rdinarily take place only m an al~alll1e so~ution,. ~lthough the r~ducll1g ~gents are elth:; neutral or,~c1d. Thus m addition to the active reducing agent (or deve.lo'per ), the s?lu,tion must contain an alkaline substance as well. This 1Susually SOdlU.111 carbonate, although other alkalis, such as the hydr,o~ides (ammonia, for example), borax, etc. are als~ used, As the activity of t?e ~evcloping agent is somewhat proportional to the amount of alkal~ pI~sent, and as the more rapid the reaction, the greater the contra~t, It W11lbe apparent that, other th.ings b~ing equal, greater contra.st m the neg,:tive can be secured by mcreas1l1g the percentage of sodium ~arbonat , and greater softness by decreasing it. Sodium carbon~te 111 the developer also acts to soften the emulsion so that the solution can penctI ,lte it more readily. , ' Examination of published formulas will show that practJ~ally all developing solutions contain, in addition to the carbon~1te, sodium sulphite as \V '11. ~Ihis is add d ro pr .v 'm t h dev'I,op1l1g agent from rapidly ai>sorhing my rcn [rum the air, ()xy reil \VIIInot only reduce

PHOTOGRAPHIC

PROCESSES, MATERIALS,

AND EQUIPMENT

THE DARKROOM

257

the solution's strength, but will disc 01or it to adark brown, which can produce staining of the gelatin of the emulsion. A chemical which will prevent this is primarily a preserving agent. Sodium sulphite possesses ~ greater affinity for oxygen than the reducing agent, and hence keeps It from the latter. As a rule, those developers keep best which can take the largest percentage of sulphite without interference with the reducing action of the developing agent. . Th: other important constituent of developing solutions is potassium bromide, This is added only in small amounts and is termed a restraining agent. When it is pot present, an emulsion will be more or less reduced, or darkened, even though it has not been exposed to any light. Such darkening is called chemical fog. It has been found that when pota~sium bro~i.de ~spresent in the solution, it restrains the general reduction and lI~ItS It to those portions which have actually been exposed to the light, so that the amount of darkening can be made to correspond to the degree of light acting on the plate. T~1Uswe see th.at (,with few exceptions) a developing solution must consist of a com!:>manon of. four chemicals: the reducing agent (or developer), a.n acnvato.r (sodium carbonate or other alkali) , a preserving agent (sodll1I? sulphite), and a restraining agent (potassium bromide). ~he reducing agent can be anyone of many which are commercially a,;aIlable for the pu:P?se, or, often, a combination of two which possess d1.ffe~ent cha~a~tensn:s. By changing the proportions of the various constituents, It ISpossible to secure a wide range in the degree of contrast or softr:ess. This process may apply not only to the development o.f the negatIve~ but to the making of the finished paper print (or positive) as well, since the latter process is identical in principle with the development of the plate. To enable photographers to secure the best results, the manufacturers rec?m.mend certain developing formulas for both plates (or films) and pnntlr:g papers. It is a good plan to begin by following the recornmendations of the manufacturers until one becomes thoroughly familiar with the principles involved. The Chemistry of Fixation N.egatives retain i~ the emulsion undeveloped silver salts where only partial or no reduction took place; if these are not eliminated, r till 'tion will continue to occur when the negative is exposed to light. ot' only would such subsequent reduction be fatal to the negative, sine' it

would ultimately become completely black, but the presence of the unreduced silver makes the emulsion relatively opaque, whereas to produce a positive print, it must be transparent in unexposed areas. For these reasons, before the developed negative can be exposed to the light it must be placed in a bath of hyposulphite of soda (commonly known as " hypo "). In this chemical all the undeveloped silver salts are dissolved out; the process is termed fixation. The reduced silver is not affected. Although plain hypo will accomplish the fixation, fixing bath formulas usually call for additional chemicals. In the so-called" acid hypo," these are generally sodium sulphite, acetic acid, and pot~s.sium alum. The acetic acid is employed to neutralize the alkali ll1troduced. by the developer; the sulphite has a double function - to preserv~ a,gall1st oxidation (as in the case of the developer) and thus prevent discoloration and also to prevent a chemical reaction between the acid and th.e hypo, which would cause free sulphur ~o sepa.rate. out. TI:e alum IS used to harden the gelatin of the emulsion so It will not fnll or fray during the subseq uent washing it must receive. The second stage of the photographic process - making the final positive print - is identical in principle with the production of the negative, so that fixation by means of hypo is required her.e also. .. Before considering the actual technique of developing and pnntIng, we should consider the equipment, materials, etc., required for the various stages .of the work.
EQUIPMENT

(I)

The Darkroom

All development must be carried out .ir: the absence of any light to which plates or printing papers'are sens~tlve; some .form of darkroom is therefore essential. When only occasional work ISdone, ?arkr.ooll1s may be improvised in a closet, kitchen,. or bathroom, especially If developing is done at night 'when no daylight need be excluded: Su~h a solution of the problem is a help to the amateur who otherwise might feel that the obstacles to taking up photomicrography were too numerous for him to overcome. Where more serious work is contemplated, some form of per\11anel~t darkroom will prove valuable. It can often be constructed 111 an arnc room or the c llar. Although it need not be large or laborarcly fur rushed, the nearer it 'all conform, ill !!cncr:t1, to the ideal darl room, ;\S

PHOTOGRAPHIC

PROCESSES, MATERIALS,

A TD EQUIPMENT

THE DARKROOM

259

required .tor semic?m~ercial purposes or work on a larger scale, the more satisfactory It will be found to be. The requirements for an ideal darkroom are: .(I) Th~ complete elimination of all extraneous light, combined with electncal outlet sockets for connection of lights, apparatus, etc., wherever required. (2) Ample room for carrying on all the work which must be performed in it. At the same time, it should not be so larze that needless steps must be tal~en du.ring the. various development pr~cesses. (3) A large sink with runnmg cold water (hot water is sometimes desirable, but is not essential). (4) Ample bench room for the development stages. The bench should be reasonably close to the sink. (5) Shelves for storage of developing equipment, chemicals, and photographic supplies. (6) Suitable means of guaranteeing against inadvertent opening of the door or entrance of light during periods when plates or paper could be accidentally fogged and ruined. (7) If tl:e temp~rature of the room is likely to be raised unduly because of its ~ocatlOn or confined condition, a ventilating system is almost a necesslty. . ~s most d~rkrooms must be constructed with regard to space limlta~lOns, all will naturally vary somewhat in the way in which they are adjusted to these restrictions. It is, therefore, useless to illustrate the layout of.a theoreti~ally p~rfect arrangement. Furthermore, every worker. will evolve 1115 own ideas as to the best design. If the requirements listed are borne in mind in working out specific designs, the final results should prove satisfactory. A few comments and suggestions, however, may help the worker to secure the best possible arrangement. . By far the best .method for insuring safety against accidental openmg of the door, either by the operator himself from the inside, or by someone else. from the outside, is the use of a labyrinth entrance. Wh~re there ISample room, the construction is probably no more expensIve than that of a door; certainly it is less costly than a double-door arrangement. . A ~ayout of such a l.abyrinth is shown in Figure I5 I. Where the light m the outer room ISexcessive, a movable dark curtain at the outer entranc~ may be req~lired. With this type of entrance on need never' fear ~ccIden~al foggmg of a box of plates or paper, or spoiling of a neganve dunng development, by SOI11COI1 unwirrin r\ cnrcrin the

darkroom. On the other hand, free passage in and out .of the ?arkroo.m is always assured. This system also allows a better circulation of air, . without resort to artificial circulation . Opinions vary in regard to the finish of the ?arkroo~ inte~ior. Three different colors are in common use - white (or light tint), black, and green. The argument for white is that if the safelight is really safe, whatever color is in use, 'white walls enable one to see better, and yet create no danger of fogging a plate or film. Green has
Slidino
opaque cU1"tain

on Rod_

Darkroom:

FIG. 151.

Labyrinth Opening into Darkroom

the advantage that the eye ~s esp~cially se?sitive in this region, an~1 hence can utilize licht of an mtensIty to which most plates are msensitive, and the room appears much brighter than it actually is, photographically speaking. On the other hand, there are so many plates and .films in current use which must be handled in absolute darkness, that the operator, is compelled at times to know how to cond~ct all operations without seeing what he is doing. Hence the questlon n~~lrally arises, "Why not carry on all operations under an a~solute ml11~m~lIn of light and be on the safe side, in every kind of safehght, by pa111tJl1g the interior of the darkroom black? " Where a labyrinth entrance is use~, this at least l?"1ust b~ .finishcd in dead black; further, unless a curtain ISalways used 111 a~dlt1on, a dead black finish is safer for the entire darkroom. As any light, however diffused it may be, which can enter by repeated reflection thr~)llgh the labyrinth, possesses rays which will fog any plate or ~lm, I1ghr~ colorcd walls in particular arc unde~ira.blc. Where black IS not used under rh s conditions, th gr n filllsh ISpr ~ l,-ablc.. . \ hen spat' . in the darkroom is a f;lt'{()~" It IS somcnmcs 1I1lPOrt:lIlt to know \ hat apparatus can la.' uSl'd (lUtSHIl' the darkroom and what

260

PHOTOGRAPHIC

PROCESSES,

MATERIALS,

AND EQUIPME

APPARA"

(11

must be inside. One operation which can be done outside is the final washing of prints and their subsequent handling, squeegeeing, drying, trimming, etc. The drying of negatives can and usually should be done outside, as the humidity in the darkroom may be high and cause slow drying. When an outside room can be darkened by means of black shades or the work can be done at night, enlarging can be done

(2) Apparatus The amount of apparatus required for dl'vl'l0l)ment and printin processes is determined largely by the worl to be ( one, the space avail
I

FIG. 153, Enameled Tray for Developing

and Fixing

able both for using and storing it, and the size of the investment which can be devoted to it. On the one hand, the minimum, 'where work / must be done in an improvised darkroom and everything packed away out of sight afterward, can be held to a safelight, three or four devel-

FIG. 152. Eastman Darkroom

Safelight

outside the darkroom. This is often a big help, especially when the enlarger is of considerable size. In general, as one stands while carrying on the various developing and printing operations, the height of the shelf should be suited to a standing instead of sitting position. This leaves considerable room underneath for shelves and storage space for trays and other equipment. Plates and materials liable to damage from solutions or w tting preferably should be stored in shelves above the b n .h, as accid 'n!s may allow seepage of solutions to storage places under the h in '11.

FIG, 154. Developing

Hanger for Cut Film

oping trays (or a developing tank), and a printing frame. Standard developers and acid hypo are available in tubes and packages whi .h require simply mixing with proper amounts of water for imm xli.n c use, Thus, cquipm nt I' .quircd for mixing one's own solutions and storing them in <Juall!il y is IIl1lH.'CCSS:lr

PHOTOGRAPHIC

PROCESSES, MATERIALS,

A'D

EQUIPME

APPARATUS

26.~

At the other extreme is the completely equipped darkroom, with every device for expediting the work. Between these extremes lies a wide range where one may choose items of equipment as needed. Even though the start is made in a small way, growth can continue until facilities are ample to handle any problems one is likely to meet in photomicrography. " Some pieces of apparatus required for commercial developing and printing are not necessary for strictly photomicrographic work where only glossy papers are employed and retouching of negatives is not ordinarily permissible. Furthermore, it is hardly likely that mass production either in the development of negatives or the making of prints will ever be required of a photomicrographic darkroom. With these exceptions, the necessary equipment will not differ greatly from that employed for commercial photography. First on the list of indispensable equipment is a suitable safelight. Such lights are available in several types, according to the specific purposes for which they are required. The name" safelight " must be flexibly construed, since a light which is perfectly safe for some work is not necessarily safe for others. Whereever limited light of a restricted color region must be employed, it is desirable that the maximum brightness consistent with absolute safety be used. 0 one light, therefore, 'I will serve for all purposes. F T I f PIate or Cut F m <JG. 155. an {or This requirement is met in welldesigned safelights by the use of removable color screens which provide proper transmission characteristics. An inexpensive form is the Eastman Safelight pictured in Figure r 52. The changeable screens (also designated safelights) are 5" x 7" in size. Number 0 is suitable for slow plates (e.g., process) and for bromide enlarging papers; #r for non-color-corrected plates of any speed; #2 for orthochromatic emulsions not sensitive to red; and # 3 for medium-speed panchromatic emulsions. High-speed panchromatic plates and films must be handled and developed in complete darkness. For those who can afford a more expensive safelight box, Fastrnan makes a model which has, in addition to the safelight, an additional opening (normally closed) which contains a ground-glass diffusing scree.n, useful for examining negatives after fixation.
I

Less expensive forms of safelights, for attachment to electric light sockets, are available as overhead lights. F or the printing processes (enlarging papers excepted) one of the best lights is an amber-tinted bulb. Low-wattage bulbs, available in red, are suitable for developing slow non-color-sensitive plates. When these are used, they should be the colored-glass type rather than

FIG. 156. Roll Film Developing

Tank

merely lacquer dipped, since the latter deteriorate rapidly and 111:1y soon reach a stage where they are no longer safe for the purpose in tended . . The type of equipment required for developing depends largely on whether plates, roll film, or cut film are used. For the former, trays (Figure r53) are preferable, since individual handling of one exposure at a time offers a better method of control. Plates are seldom exposed in such quantities as to 'warrant multiple development in a tank. Several trays are necessary; it will prove economical in the long run to have the proper sizes for accommodating each size of plate (or printing paper). They are available in glass, porcelain-lined steel, stainless steel, rubber, and composition. The rubber and composition are by far the least satisfactory. Tanks are desirable for cut film, to eliminate danger of scratching the film. The films are placed in hangars (Figure 154) and allowed to remain in them through the entire process of developing, lixing, and washing. Several small tanks (Figure J 55), capable of nccommodut ill r the largesr film used, will be required. Roll film is b sr handled in u developing rnnl of t hc rype illustrated in Figure 156; the preferred [onu of rnnk for Iilm packs IS (hat shown ill Fi rure 157. Orhcr al'Cl'ssoril's dcsirnhlc for tIll' dcvcloptuvnt prol'l'SSl'S :lIT: scalt,s

PHOTOGRAPHIC

PROCESSES,

MATERIALS,

Al D EQUIPMENT

APPARATUS

FIG. 159.

Immersion Thermometer

FIG. 157. Film Pack Developing Tank

FIG. 160. Eastrnan

Interval Timer

FIG. 158. Studio Scales (Eastman)

PHOTOGRAPHIC

PROCESSES,

MATERIALS,

A'D

EQUIPME

API'

1\i\IUS

(Figure 158) for weighing chemicals when mixing various solutions; an immersion thermometer, one type of which is shown in Figure 159, for determining the temperature of the developer; and an intervaltiming clock (Figure 160), for timing the development. Such a clock sounds an alarm at a predetermined time, which is set to assure proper development for the given combination of plate, developer, temperature, and subject. Trays are required for the printing processes, but if trays are used for negative development a duplicate set is seldom necessary. There is considerable latitude in the type of equipment used for printing the positive from the negative. The simplest device is the printing frame shown in Figure r6 r. These are entirely satisfactory but slow in operation, and therefore where time is a factor a small printing machine such as the Eastman #2 (Figure r62) is preferable. Still more pretenFIG. 161. Printing Frame tious printers are the Eastman Professional Model (Figure r63) and the Graflex Enlarger Printer (Figure r64)' The latter prints by projection instead of contact, so that the size of the print can be varied at will. This is a valuable feature when the photomicrographic camera is of small size and larger prints are desired. For the small minicams, enlarger models on the market designed especially for these small films are preferable. Even the photomicrographer equipped to take large-size micrographs may be called upon to furnish enlargements from occasional negatives. F or this purpose a machine such as the large Eastrnan Professional Autofocus Enlarger (Figure r 66) is ideal. It will accommodate plates up to 5" x 7". Printing and enlarging necessitate the use of a suitable timing clod capable of indicating exposure times to a second or even less. That suggested for use in making the exposure of the negative (Figure 77, page r 24) is the preferred type. Plates are washed after fixation in either ranks or individual t rn s,

FIG. 162. Small Printing Machine

(Eastman No. 2)

1'1<

I(q,

1',11111,111

1'10

of., iunul

Printvr

268

PHOTOGRAPHIC

PROCESSES, MATERIALS,

AND EQUIPMENT

J\ I' I'

1\ I lJli

FIG. 164.

Graflex Enlarger Printer

and must then be set in a negative rack for drying; therefore at least one drying rack is necessary. Where a number of lantern slides are printed at a time, several racks may be required. 'iY ashing of paper prints necessitates some form of washing device to keep the prints continually agitated in running water. For a few prints only the Eastman Syphon Washer (Figure 165) can be used in combination with a large tray. The Whirlpool washers (Figure 167) will accommodate a larger quantity of prints. Where many prints must be washed at a time, as in cornmcrcial work, the electricall y cl riven rotary washers, such as shown in Figure y68, arc ideal. For srri .rly FIG. 165. Eastman Syphon Washer photomicrographic worl , however,

Fw. 166. l':aslIlI;\n Auto-IIo us 1':lliargcr

270

PHOTOGRAPHIC

PROCESSES, MATERIALS,

AND EQUIPMENT

GLASS\v

A 1\11:

complete the mechanical equipment required for the photomicro graphic darkroom, although various other labor-saving devices will hl' found useful as one becomes more proficient in the work or rakes it lip more seriously. Among these might be mentioned a scri s of Illasl cutting templates and a circle-cutting tool for use with the tcmplnt 'S. The tool is available commercially, but the templates may have to h . . specially made. They are made of about I/ I 6" sheet steel or brass, with circular holes cut in them of the sizes desired for masking the neg ative in making the prints. Photomicrographs are preferably printed cirGul~r to give the same appearance one obtains with visual observanon. Another useful device is a retouching stand, for spotting defects in negatives and occasional blocking out of undesirable backgrounds. (3) Glassware Darkroom glassware requirements are not extensive. The m~)sr ill\portant pieces include graduates - preferably two-ounce, sn re '11
FrG. ,67, Whirlpool Washer

the use of large washers is usually restricted to research laboratories doing a considerable amount of work. Practically all photomicrographs are finished up on glossy paper, squeegeed to give the highest possible Iuster. This means that ferrotype plates must be available, and also a squeegee roller. Two types of plates are available, black-japanned iron and chromium-plated brass. The latter are better but more expensive. When only a few prints are made at a time and they are small, a hand roller (Figure I 69) is adequate. Better results are obtained, however, especially with double-weight paper and large prints, when a wringer is used. Wringers (Figure I70) may be either hand or power operated. An ordi-. FIG. ,68. Pa-ko Large Rotary Print nary clothes wringer will suffice. vVasher print t riIII111 'r (Fi rurc 171) will

FIG. ,69. Hand Roller for Squeegeeing Prints

ounce, and thirty-two ounce; a glass funnel (sixteen-ounce size) for filtering solutions; stirring rods; and a collection of bottles for the vari

FIG. 170.

Fcrrotypc Plate Wringer

ous solutions it may be convenient to keep on hand. For storing ('011 cent rated dcv 'loping solutions, the bottl s employed sh(~lIld I>'of such si'!.'s that the solut ions compl r lv fill t hem, rhus cxcludin ,. any ,lppn' ('ial>l<:.qllalltity of air and so pi'('vclltin" oxidation. Their I(,t'pill CJ\I:ditit's~lI'l''1T:ItI" enhunccd t1HTl'hy.
l

PHOTOGRAPHIC

PROCESSES,

iATERIALS,

Al D EQUIPMENT

I I11

111'1

'71

(4) Chemicals
For the amateur interested only in an occasional photomicrograph, the list of chemicals required is not extensive. Figure 172 illustrates a suggested developing kit. When all development is done with prepared developers, especially of the individual glass-tube type, and prepared acid hypo is used, these items can constitute the entire re-

quinone

for bot h ne r:11 iVl'" _11HI jlnllt., IIIHkr till' gl'Ilt't':tI dl'si runt ion oi
1I1()1't'

" ~-Q dcvcloper." Hydroqui1101le.

. l'()\ltr:lsty developer, (.'otnlllOllly .used I11 combination with mctol. By variations in th~ perccntag's oi Il\l:tol, hydroqllinone, sulphite, cnrbonutc, and bromide, almost ,Iny d 'sll'l'd dcgree of contrast can bc produced. , .Amidoi (Diaminophenol). very soft ":,orkll1g dcvcloper, lI~l'ful for bromide paper but also good for soft pnnts from harsh ncguuv 'S.

FIG,

17 , 2

Group of Chemicals and Apparatus

for Developing and Printing

FIG. 171.

Print Trimmer

quirements. Probably, however, no amateur photographer will long be satisfied with such limited facilities. Also, the cost of ready-made solutions is relatively high. The developing agents required will naturally be governed by the specific type of developer standardized upon, especially for negative development.' These may include such chemicals as " pyro" (pyrogallol), glycin, rodinal, and other recommended developers. For the beginner, however, the following should suffice: Metol. Sold by different manufacturers under various trade names such as Elon, Rodol Photon, Pictol, etc., chemically known by the somewhat high-sounding name of monomethylparaminophenol. This is a soft developer, and is usually employed in combination with hydro-

It does not require that an alkali be used with it" an~ hence has :111 other valuable property seldom apprec~at~d. s It WIll stanc.~ a la~''~l' .unount of restrainer (bromide), old pnnt1l1g papcr, usc\cs.s wIth, 01 dl nary developers, can often be made to yield beautiful whites wit hout
a t 1':\ 'c of fog.

. . . . W orkers with ,11111:Iatnrc.cameras, wtt It which considerable enlargement of the neganve IS required for prorlu in r a satisLlctory print, will do better at the start to employ ,on' of t Itv munv oommcrcial fine-grain dcvelopers on the marker for thl~ plIrpoHl': Tlll'se .omc ready pr pared and give superior results for this l:tss ()i

Fille-grClil1 developers.

\\' 0 III

rk.

add it ion to the d 'velopin r a rents, t h ' following

.hcmi 'nls shoulrl

he uvuilnhlc:

274

PHOTOGRAPHIC

PROCESSES,

MATERIALS,

AND EQUIPMENT

DEVELOPING

Al D PRI TTIN(;

TEell

'IOlH:

7.')

Hypo Sodium carbonate Sodium sulphite Potassium bromide Acetic acid

Potassium alum Borax Sodium bisulphite, or Potassium meta-bisulphite Potassium ferricyanide

These will be found adequate at the start and can be added to later as the need may arise. Among the more important additional chemicals of value in ordinary photographic work might be mentioned: Potassium chrome alum Potassium dichromate Potassium permanganate Formalin - (407'0 formaldehyde) Sulphuric acid

Developing and Printing Technique


Appreciation of certain underlying principles involved in the developing of negatives and prints will enable one often to detect reasons for poor results and frequently to improve the quality of the work. As we have said, amount of contrast can be controlled by use of different developers. The exposure of the negative also plays an important part in the nature of the finished print. A third factor is the extent of the development of the latent image. For any given combination of developing chemicals, the latent image is gradually built up at a time rate dependent upon the concentration or strength of the solution and the temperature at which it is being used. Cold solutions work more slowly and warm solutions more rapidly. The ideal temperature for development is around 65 F. Below 55 F. the action is very slow; some developers will not act at all at temperatures much lower than this. On the other hand, temperatures in excess of 75 F. tend to soften the emulsion to such an extent that a negative may be ruined unless special precautions are taken. Other unsatisfactory conditions may also result from high temperatures. Between the ranges of 55 and 75 substantially equivalent results can be obtained with most developers by varying the time of development to suit the temperature, provided that the development is carried on to substantially the ideal density. Whatever variations do occur under these conditions are largely the result of changes in the relative action of two de veloping agents in the solution, such as metol and hy lroquinon , <It

the various temperatures. Metol, for instance, continues working at 55 F. while hydroquinone becomes practically inert at aro~n? 60 F. The effect in M-Q combinations is therefore somewhat similar to a change in the percentage of the two active agents in the d~veloper. Weaker developers must be allowed to act for longer times to produce equivalent density in the negat~ve. In general, .however, strong solutions yield greater contrast, while weaker solutions tend to decrease the contrast, thus producing softer negatives. Weak developers may be weak because of greater dilution in th~ beginni.ng, or a strong developer may become weak ~hrough exhaustl?n resulting from repeated use. Also, developers which have been mixed up fo~ sO.I11C time, as stock solutions, may lose so much strength through oxidation that results are not identical over an extended period. All of these factors should be always borne in mind. . . Agitation of the developer during development hastens the actl?n. With tray development this is easily accomplished by gently rocking the tray. This is one reason for frequer~tly observed supenor results with plates, since the latter are more easily handled by tray develop ment, esrecially when, as in photomicrography, they are developed one at a time. The particular type of plate employed has an appreci~ble effec~ Oil the development to which it should be subjected. WIth any f$IVCIl plate there is a range within which the density produced by ~l1Ifonll development conditions is directly proportional to the loganthm of the exposure. This is usually portrayed graphically as the D-Iog I'~ curve (Figure ~73) . . The proportlon of the curve which IS a straight lme WIll vary: WIth different kinds of plates. The region .of correc~ exposure. IS the straight-line portion of the curve - that IS, the various grad.atlon. of tone within the object should, if possible, all fall on the straight 11I1~. In photomicrography by transmittedJight, wl~ere ~he background IS often extremely bright as compared WIth the object Itself, the exposur of the background will, with many types of plates, fall on the top curved portion of the cur:re. That is, it will be overexposed, bu thiS is not objectionable. It .IS ~he tonal l:ange. of the ?bJect portrayed which should be kept within the straight-line portIOn of the curve wherever possible. . . Sin .c there is in development, at the e act moment of b gll1l1l11g of dark ning, a zero difT rcnc .in rh' <I~tllalamount. of .onrrast i.n the ne rutivc, between those port lOllS which have re 'CIVCt!the muxuuum

PHOTOGRAPHIC PROCESSES, MATERIALS,

AND EQUIPMENT

DEVELOPING AND PRINTING

TECHNIQUE

277

amount of light and those exposed to the minimum light, the function of the developer as regards the time factor is gradually to build up the difference in density of the various parts of the image. This difference is designated the development factor or ga711111.a ('(). It is shown in its relation to the slope of the density curve in Figute 174. Study of this graph will reveal that the ratios of the straight portions as regards the densities are the same in both fractional development curves but the actual differences are greater as the value of gamma increases. In other words, the steepness of the curve increases with increase in
2.0 I.B
1.6

packed with each box giving the proper development time for low, normal, and high contrast for the emulsion number of the particular box, with the recommended developers. This is a valuable aid toward securing ideal results, since the development can be done on an exact time and temperature basis, even in total darkness. . Some workers like to observe the density of the negative by holding It up to the safelight for examination by transmitted light. This is a poor practice, since fogging can easily occur. A safer method, if an examination must be made, is to observe the image build-up on the
2.0 1.8 1.6

'Y.8

1.4 >- 1.2


0-

1.4
1.2 1.0 .8 .6
1.5 .50 LOG 1.50 EXPOSURE 2.5 3.5

Vi 1.0 ~ .8

c .6
.4 .2

.4 .2 1.5 .50 1.50 2.5

FIG. 173.

Exposure Curve.

D-Log E Curve

FIG. 174.

Graph of Development

Time

gamma, indicating an increase in the contrast of the negative. This contrast continues to build up to a maximum designated gamma infinity ('(cd, after which further development causes a decrease in the contrast. Development beyond '(00 is called ouer-deuelopment. Full development is usually considered to be reached at around 0.8 '(00. Th~ actual value of gamma infinity is determined by the type of ~mulsI~n. A soft plate has a low value. Usually this type of emulsion IS relatn~ely ~ast. The slow process type of plate possesses a high gamma ~nfi11lty. In o~der. to reduce the actual contrast normally present m such plates, It will be apparent from Figure 174 that, assuming a full exposure has been given, reduced contrast to almost any deg~ee. c~n be obtained by giving a shorter development. In domg this It. IS often useful to employ 'weaker developing solution s? as to provide a greater latitude in the development time. The deSIred gradations in the negative are more easily secured with slower development. . From the practical standpoint, most developer formulas have th ' time o~ de~elopment at a given temperature stated so as to yield all approximation of 0.8 '(00 for average types of plates. Some makes of plates (e.g., Wratten M and Wratten Panchromatic) have .ards

back of the plate. When the high lights (i.e., the most dense portions) begin to show through, it is an indication that development has taken place through the entire depth of the emulsion in these portions and there is little chance of extensive under-development. Another method is to observe a decided darkening of the shadows (i.e., those portions receiving the least light) on the front of the plate before considering dev~lopment to be complete. Of course, variations in individual subjects considerably vary the application of either of these methods for completely satisfactory results; also, the emulsion of the plate itself is a factor. Experience, however, soon establishes the standards to go by. When experience is gained by repeated observations of the appearance of negatives under the time and temperature method, correlated with the final results, it is a big asset. . Should extremely dense or extremely thin negatives consistently rcsult in spite of faithful application of the time and temperature method, the trouble almost always can be traced to over- or under-exposure. To correct such a fault, bear in mind that the effect of the xposlIre Iim . on density varies in gcol11 .rric ratio, hen .c the first stcpS should h . tul ell hy Ilalvl11g or dOllbling lilt' ('XPOS\II'(' rime, as may be ne cssary.

278

PHOTOGRAPHIC

PROCESSES, MATERIALS,

AND EQUTPMENT DEVELOPING AND PRINTlNG TECH IQUE

279

If the correction is still insufficient, the new trial time must be again halved or doubled. When development is comple~ed, ~he plate (or film) should be rinsed in water acidified with acetic acid (see formula at end of chapter) to stop development. and neutralize th.e alkali ~f d~e develo\er so that the hypo solution will not be cont~mI~1ated with It. .A few seconds' rinse usually suffices. The nega:Ive IS then placed ~n the hypo bath. It should not be examined by white light until the milky appea~ance of the emulsion has completely disappeared. The total time m the hypo should be about twic~ that requ.ired for the appare.nt removal of the unaffected silver salts (i.e., the milky appearance), 1~1 orde~ to assure complete fixation. The negative can the.n be washed m TImnm$ water until all the hypo is eliminated. If. runn111g water from. a tap ~s allowed to flow directly over the negative, ten ~o fifteen mmute~ IS ample but when the negative is in a tray or container where a considerable' body of water stands, the. time s?ould be at least one-.half hour. When running water IS not available, It should. be washed 111 about a dozen changes of water in a tray, ov~r a period of about .an hour. After washing, the negative should be lightly ~ubbed ov~r WIth a tu.ft of wet absorbent cotton, rinsed, and set aside 111 a negative rack or m hanging clips to dry. .' . . Paper positives fro:n negatives a.re made by usmg the saI~e baSIC principles as for negatives. ~h?tomlcrography does not call for the superabundance of effects, pnntmg processes, types of paper, etc., ~sually employed by the professional. or amateur photographer, sJ[~ce practically all prints are made on white glossy paper, squeegeed to gIve the highest possible gloss. .. . . The paper stock is furnished m thm, or single-weight, .and .heavy, or double-weight paper. The latter, though more expensIve, ISpreferable, and after being once used :.vill proba?ly never be abandon~d ~or single-weight prints, for photomlcrograph~c purposes. Th~ maJonty of photographic paper manufacturers fur11l~hglossy paper m as many as five or six degrees of contrast, usually designated by numbers. In the Azo (Eastman) and Apex (Defender), two of the most commonly used papers, # 2 is cons.idered the proper grade for l1?fmal negatives and the expert photomicrographer should expect tI~ISpaper to suffice for at least ninety per cent of his :work. ~umber. 0 ISvery sof:, for use with extremely contrasty negatlv~s, while # 5 IS an cxrr III contrast paper, suitable for negatives devoid of cO~ltr<l!\t. . The intermediate grades e.nable Ol:e t? .worl sltgh~ I)' Oil.t,he sO,ft.(~I, cont!asty side, as may be desired for individual ncgut Ives. I he III gl tl

of contrast can also be controlled to some degree by the developer employed, the exposure and time of development, just as in the case of the negative. With normal negatives, the best results are obtained with a soft, slow-acting developer, rather than a contrasty one. The exposure time for a given negative and constant light source will be found to vary greatly with the different grades of paper, #0 being the fastest and #'5 the slowest. There are some differences in the speeds of the same contrast grade of paper as made by different manufacturers, and, also, the degree of contrast for a given number may vary somewhat. Because of these conditions, it is desirable either to standardize on one make of paper, or establish comparative reference data as to contrasts, printing times, etc., for different brands so that recorded printing data for a given negative can be translated in terms of any of the papers likely to be employed. Printing may be done in either of two ways - by contact or by projection. In Contact printing the emulsion surface of the paper is placed directly against the emulsion side of the plate or film, and must be held in contact under pressure while the print is being exposed. The sim plest apparatus provided for this purpose is the printing frame (shown in Figure 16 I). It should be needless to remark that loading of the printing frame should be done only in the presence of a light which is not capable of fogging the paper, and that care 11lUSt be taken to ha I.' ~very bit of sensitive paper (such as the box or package of unexposed paper; prints already exposed, but not developed; and prints in the developer undergoing development) thoroughly covered before turning on the light to expose the print. When printing is done in a frame, in making the exposure the distance between the lamp and the printing frame should be maintained at a constant, fixed distance. This can be done as illustrated in Figure 175 The frame should be held at right angles to the central beam from the lamp and at the same time should be sufficiently far away that the light paths to the outermost corners of the print are not appreciably more than the distance to the center of the print, as otherwise uneven illumination will result. This means that a large picture must be h Id farther away than a small one to secure even illumination over the elltire area of the print. As the light intensity varies inversely as the square of the distance from the light to rh negative, when t he printing frame is 1ll0V.d twin' the distance away, the (;xposur' 1I1USt bl' in Te:1S.d by four times for un cquivnlcnt exposure. 111 or.dl'!' to product, a circular pictul'l' UI'1'OIIlHkdhy :l white l)(Jr

PHOTOGRAPHIC

PROCESSES,

MATERIALS,

AND EQUIPMENT

DEVELOPING

AND PRINTING

TECHNIQUE

der, a circular mask of the desired size, cut from thin black paper, should be placed between th~ negative and paper, in.th.e prin~ing fran:e. Circular prints are usually trimmed square after finishing, WIth a white border of from one-quarter to one-half inch margin of white on the sides. This allows the cutting of test exposure strips from the long dimension of the paper. For instance, if the paper size is 5" x 7", the

FIG. 175. Exposing Print in Printing Frame

maximum square would be five inches, the circular picture about 4t" in diameter. A one and a half inch strip can be cut with safety from the seven-inch dimension of the paper, to provide a test strip for determining the proper exposure time. Negative envelopes are available for storage and protection of .the negatives. These provide an ideal place on which to rec.ord the printing data. The make and grade of paper and exposure time should be recorded, as 'well as the developer, if different ones are employed which require a variation in the exposure time. . Small printing machines (or boxes), such as illustrated in FIgure 162, are a great convenience, not only because of the increased speed of operation, but because the rrinting light is completely enclosed, so .tl:ar there is no need for covering up the box of paper or other sensitive material every time a print is exposed, as must be done with printin r frames.

Printing by projection is desirable only when the size of the finish d P!Int ISrequ.Ire~ to be .larger than the negative. Such printing calls [or either a proJectIon pnnter such as the Graflex model (Figure 164) OJ' aI?-enlarging machine. Where the enlargement exceeds a couple of diameters, the ordinary contact paper is not usually satisfactory be cause of the inefficiency of the illumination systems employed, and hence bromide enlarging papers must be used. These are extreme! fast as c?mpared to contact printing papers, and therefore must he handled In more subdued light, such as the safelight #0. Bromide papers ca.11 fo! weaker developers than contact papers, and the development time ISlonger. Whatever paper is used, it is preferable to employ with it one of the specific developers recommended by the manufacturers. . Prints ~hould be rinsed in an acid short-stop bath before being placed In the fiXIng bath. The latter should be clear and fresh in order to ,ISsure freedom from staining, and complete fixation. As paper prints do not show any change in the fixing process (so apparent in the case of the negatives), the only way to be sure of perfect fixation, without which the prints would not be permanent, is to rely on the solurion being of suitable strength and acting for a sufficient time. If unused pieces of old plates are saved, the strength of a hypo solution can he tested by observing the rapidity with which the milky emulsion clears when placed in the hypo. If it takes more than a couple of minutes for a marked change to occur, the solution should be thrown away and a fresh one used. Ordinarily about fifteen minutes in the hypo is ample for prints. They should be well rinsed on removal from the hypo bath and, when the batch is completed, washed in running water for about an hour. The next operation is the. placing of the prints on the ferrotypc pl~tes, face down, directly out of the water, without draining. Chromium-plated plates rarely require preparation other than thorough washing in plain water, but the japanned plates should be waxed be fore being used, and at any subsequent time when evidence of sticking appears. Consult the formulas at the end of the chapter.for the wax s~)lution and method of applying it. The ferrotype plate should he 1111sedin cold water before being used, and as much water as it will rernin be allowed to stand on it. When the print is on the f rrotype plate, it must be squccgccd into p rfccr conta .r, irh r by J'unning rh rough a wringer or with the hand roller; then all surplus w.uvr must be wiped off and the pl.u c

PHOTOGRAPHIC

PROCESSES, MATER1ALS,

AND EQUIPlVIENT

REDUCTION

AND I TTENSIFICATION

set aside for the print to dry. When only partially dry it will adhere tightly to the plate as though glued to it, but when completely dry should fall off of its own accord. If it does not, a little loosening at one edge will enable it to be easily stripped from the plate, if the waxing has been properly done. It then remains only to trim the edges of the print to the proper size and the job is done. An added convenience for the future identification of the print is a rubber stamp for use on the back of the print, with the photomicrographer's (or company's) name, and space for the negative number, subject of picture, and magnification.

Reduction and Intensification


When a negative is too dense to allow a satisfactory print to be made from it, or, on the other hand, is thin and Jacking in density, it is sometimes possible to improve its printing qualities by reduction or intensification. Reduction is a chemical process which dissolves out a portion of the silver deposit in the emulsion so as to make it less dense. Intensification builds up a greater metallic deposit, either of silver or some other element, on that already present, to provide greater density. Although in general the photomicrographer will find it more advantageous to throwaway a negative not up to standard and take the micrograph over, rather than attempt to salvage the poor one, there are occasions when this may not be possible. F or this reason familiarity with the methods of reduction and intensification is desirable. egatives that are too dense result from either over-exposure or over-development, or a combination of both. Since both excessive over-exposure and over-development tend to reduce contrast, such negatives are apt to produce flat prints, although this is not always true of negatives benefiting by reduction. In the majority of cases the obvious reason for reduction is an exceedingly dense negative requiring a printing time running into minutes, instead of five or ten seconds for an average-density negative. On the other hand, cases arise where the object photographed is naturally contrasty in some of its component parts. It should have been photographed on a soft plate, with a full exposure and a development on the short side, but instead, may have been taken on a contrasty plate and received full development. The result is a contrasty nega-

tive requiring a long printing time to give detail in the dense portions, while the remainder of the negative yields a fine print with a normal printing time. Partial correction of such a negative through prop 'I' reduction may improve it. o single reducing method will suffice to meet every condition, as three possibilities of improvement exist. F or instance, (I), with a negative which yields a print of proper contrast but requires an excessively long time to print, what is needed is proportionate reduction so that while the negative is made less dense, the contrast is not changcd; (2), a dense negative producing a flat print requires reduction which at the same time will increase the contrast; while (3), one which is xcessively contrasty must be reduced in such a way that the contrast will be diminished. Many different reducing formulas have been proposed to meet these conditions, but those given at the end of the chapter, together with their characteristics and methods of use, will be sufficient for the av erage photomicrographer. As a matter of fact, Farmer's reducer will probably be the only one required for ordinary work. Thin negatives result from either under-exposure or under-develop ment. In ordinary photography under-exposure may occur from sheer inability to take a picture under conditions where it can be fully cv posed. Combinations of available light, emulsion speed, and movement . of the object (which determines the speed at which the exposure must be made) are sometimes fixed to such extent that a fully exposed nega tive cannot be obtained, and a retake under more advantageous .ircumstances is impossible. The only recourse is to build up a fairly good or passable negative by intensification. In photomicrography one seldom encounters similar conditions. If an under-exposure 0 curs, it is usually the direct result of miscalculating the proper exposure time. Therefore, thin negatives which result from under cxposure should be taken over rather than corrected by intensification. The possibility of intensification of a negative lies in the ability of the silver deposit in the emulsion to build up further metallic density in substantially the same proportions as already present. In areas where no silver exists in the negative, as usually happens in underexposure, there is nothing to build upon, so that intensification, thouuh it Imy produce contrast between light and dark portions, will not provid ' desired d tail ill the dark areas. The siumt ion is quit ' differ .nt when thinn 'ss of the negativ ' rcsultx fl'Ol1l under development of a filii) npos(:d plate. 11 '1'<:there will be

PHOTOGRAPHIC

PROCESSES, MATERIALS,

AND EQUIPMENT

THE

MAKING

OF LA TERN

SLIDES

silver present in the proper ratios, and if it can be built up by proportionate additions of silver or other metal, just as though developm~nt were being ~ontinued from the point where it was stopped, a fairly good negative may be produced throuoh intensification. It shou.ld be borne in mind, however, that in many cases where intensification could be accomplished with profit, the simpler method may be to retake the exposure and develop it properly rather than bother with intensification which may, or may not, give satisfactory results. A few formulas and instructions for intensification are included at the end of the chapter for the benefit of those who may desire to try t~1em, or for those rare cases where it is impossible to retake the negative. The Making of Lantern Slides Because of the ~cientific an? educational value of photomicrographs, tl:e photomlcrograpl:er IS f~equently called upon to produce lanter~1 slides from som~ of his negatives. Apart from a few additional steps m the p~ocess, thl~ class of v,~ork does r:ot differ fundamentally from the making of ordinary negatives and pnnts. . The ~tanda.rd Americ~n size for lantern slides is 3t" X 4" (the longer dimension bemg the honzontal one, as the slides mount in the projection lantern) . * The lantern slide is a positive transparency, printed on glass, instead of or: paper. Manufacturers supply lantern-slide plates of the stand~rd size. They are very fine-grained slow emulsions, and can be had m at least thre.e degrees of contrast - soft, medium, and contrasty. For properly timed and developed negatives, the medium or normal plates are the best. The picture size, to utilize the most of the plate area, should be about 2 iN dia~eter for a circular print, or 2 t" x 3" for a rectangular one. In unusual instances these dimensions can be increased slightly, and, of course, the~ can b~ as much under as desired, but at the expense of projected picture size when full-size pictures are also shown on the same occasion, . There are three possible methods of printing lantern slides, dependmg upon the size of the negative and the area to be embraced in the
The standard English size, also quite commonly used in Canada, is 3~" x 3l". Manufacturers of lantern slides supply holders which will accommodate either, and special holders are made which will take both, indiscriminately.

lantern-slide picture. The simplest case is where the lantern-slide print is to be the same size as the negative, since this can be aCC0111 plished by contact printing. This is preferably done in a printin r frame, as the intensity of the light in a printing machine set up for gas-light paper is usually far too great to allow of proper control of the exposure. Generally about one to three seconds exposure through a normal negative 3 feet from a r o-watt lamp suffices. The negativ . need not necessarily be the same size as the lantern slide for contact printing. If small prints are satisfactory for projection, films fron: minicams can be made into standard-size lantern slides by contact printing. Care must be taken, from the aesthetic point of view, that the film be properly centered and aligned on the lantern-slide plate. Should the negative be at all dense, the use of a black paper mask around the negative is desirable, not primarily to stop the blackening of rh plate around the picture, but to obviate almost certain halation around the edges. Lantern slides can be made by contact printing from even the lnr r est plates (e.g., 8" x r o") where only a specific area is required to ht, shown and can be included within the confines of the lantern-slide picture space . Usually photomicrographs on minicam film which are to be made into standard-size lantern slides should be enlarged to the proper di mensions. This enlargement can be done easily in any of the standard . minicarn enlargers, merely substituting the lantern-slide plate for the bromide paper. If an intense light is employed in the printer, suitable for enlarging on gas-light papers, it must be cut down to the bromide paper range to allow sufficient latitude in exposing the plate. The third condition obtains when photomicrographs on large plates must be reduced to lantern-slide plate dimensions. For this worl some form of copying camera must be used and an illumination bo: provided for uniformly lighting the negative from the back. Often the photomicrographic camera can be utilized for this purpose, or th copying may be done with any camera which will accommodate a 3A" x 4" plate and which possesses a bellows length ample to provide focussing flexibility for the lens employed for copying. For rcducin r .1" X 7" plates (4l" circle) to lantern size (2t" circle) a 6-i11Ch lens is satisfactory and the reduction is roughly to three-fifths of the original. Sine' the s 'ale of a rrallspar 'lle)' is nor limited ro the same .xrcnt as a papl'r print, iI 1110recontrast y development is p .rmissihlc and giv(,s

286

PHOTOGRAPHIC

PROCESSES, MATERIALS,

AND EQUIPMENT

to the projected picture a snap that is pleasing to the eye. Development should be complete or the picture will appear flat. One must not be misled by the appearance of the image while the plate is still in the developer, for fixation changes the values of the tones materially. Development until the picture appears far too dark (if it were a paper print) 'will be found to give a much better result 'when the slide is projected. Development, rinsing, fixation, and final washing of lantern slides do not differ from the standard process for plates. When the plates are dry, the next step is the mounting and binding operation. For this, three additional supplies are necessary. These are: a mat made from opaque paper of the same size as the lantern slide and with a hole cut in it of the desired size and shape to reveal only the picture area; a lantern-slide cover glass, which is merely a clear glass of the same size as the lantern slide; and a piece of binding tape for passepartouting the glasses together. Mats can be purchased with various-sized openings, but when only occasional slides are required and the pictures are not such as to be accommodated by standard mats, they can be made of opaque paper as necessary. Circular openings can be scribed with a pencil compass and cut with small scissors, or a circular print cutter can be used. For a large quantity of circular mats a steel punch of the desired size can be obtained. Square and rectangular openings can be cut out with a safety-razor blade by first making a cardboard template of the desired size and shape and cutting around it. If desired, thumb markers, the photomicrographer's name, and subject of the picture can be typewritten on paper inserts and included within the slide before binding, or they can be pasted on the outside later. The glass covers are obtainable from any supply house dealing in photographic supplies. These suppliers also carry the binding tape, which comes in two forms, continuous rolls or cut pieces I S" long. The binding is usually one-half inch wide, and gummed on one side. It is applied by starting at one corner and doubling the corners over so that it runs continuously all around the slide. In order to hold the two pieces of glass together while being bound, spring clips mounted on a baseboard are available at small cost. In assembly of the mat and cover to the positive, care must be taken that the emulsion side of the latter is on the inside, since the major purpose of the cover glass is to protect the film against scratching. The introduction of miniature film in recent years has resulted ill the development of small machines suitabl for" projtt:ting this sin'

of transparency. If photomicrographs arc to be made in.to lant '1"1\ slides for use in these miniature projectors, the only change 111 the pro cedure lies in the size of lantern-slide plates employed (2" x 2") .md the greater reduction necessary when any photomicrographic OUI Iir larger than a minicam is used. . With ordinary lantern slides it is important that they be Inserted in the projector properly, as otherwise they ~ill ~e upside down or .reversed left and right. To assure proper placing 111 the lantern, which actually means placing them in inverted, it is cus~omary t.o place an indicator, known as the thumb mark, on every slide. ThIS can be a dot, circle, star, or even a small square of paper pasted on the outside. It is placed in the lower left-hand corner as one holds the slide before him and views it as it should appear on the screen. . . In placing the slide in the mach.in~, the oper.ator grasps the slide In such a manner that the thumb of hISnght hand ISover the thumb marl as he faces the screen, and drops it into the lantern in this position. 1~1 other words, it is then in the upper right-hand corner, and hen" IS inverted, but is not reversed from left to right as long as the operator is facing the screen, that is, is viewing the picture as if he were holdin r it in front of him. With many photomicrographs, it is unimportant how they arc viewed, but for the sake of uniform practice, it is desirable that every slide be provided with a thumb mark. In this 'way a lecturerbecomes .accustomed to the exact appearance of the picture on the screen a~ld can readily locate a particular object to be pointed out. Then ~gall1, there are microscopic objects which should always be show~ 111 r~1e proper position. To rev.erse them .would seem to the expert blOl<>glst just as serious as to project the picture of a tree or a h.orse upside down and attempt to explain it to an appreciative (?) audience. FORMULAS The following formulas are provided largely as. a ready refer n~e list of those published and recommended by various photograph I ' plate and paper manufacturers .. They.are by no means the only on s which arc in use or known to gIve satisfactory results, but should be ample to meet the average photomicrographer's requirements .. Sine stri .rly photomicrographic work d~)cs n.m call for IOllll1g 'f Iccts, various proc 'ss's widely used hy I,h 1'1.rorial photographel:, and unusual techniques, all Ionuulus pcrruinin r 10 II~(.'s<.: have. he .n ()Illltt,l'll. III addition, Ihl'IT an' 1l1:1II)' [ortuulns l'lllpl()yll\~ s]llTlal dt'vt\()]llll'

288

PHOTOGRAPHIC PROCESSES, MATERIALS, AND EQUTPMENT

FOR1VIULAS

agents which are of considerable :ralue in t~le general photographicfield, bu~ unnecessary for photomicrographic purposes, which have been omitted. These can be found, if required, in almost any of the handbooks and more ostentatious works on photography. The development of color transparencies such as the Lurniere, Agfa, and D~fay plates ~nd film.s requires specific developers, the formulas for which are furnished WIth each box of plates or films. The same is true of other color processes which might be used in photomicrography, such as the Defender Chromatone Process. Certain procedures and precautions apply in general to the making up of every formula, and hence it is well to understand these at the o~1tset. Among these the following are of importance - sometimes VItally so, and at oth.er times may be considered only as recommended practlce; but one will always be safe by assuming them to be factors 111 every case, not only in formulas given here but those derived from other sources. ~I) The. water used for mixing solutions should be pure. Theoretlcally, this would mean that only distilled water could be used, and hardships often result frO~11 su~h .a ~equirement. Actually, most tap water can be used, especially If It IS filtered to remove iron or suspended matter when present, and when boiled and filtered (on cooling) if the water is that type known as "hard." Hard water can be recognized by its tendency ~o neutralize the effect of soap on the hands. Calcium and magnesmm salts are present in such water and can cause m?ch trouble. Boiling tends to precipitate them. With these e~ceptlOr:-s, good clear tap water should suffice; however, if trouble ISexperienced, look carefully to the water used. (2) Only ch~micals kno~n to be. chemically pure or prepared by one of the chemical compal1les especially engaged in the manufacture of photographic supplies, should be used. I~ some cases the same chemical (e.g., metol) is known by a specific trade name with each manufacturer. Therefore, in any formula calling for such a substance by one name, it must be understood that the product of another supplier can be substituted with safety, if one first makes sure of the actual identity of the chemical involved. (3) In making up a formula of any kind, crlwcry s follow the order in which the chemicals arc given, unless specific instructions arc added :IS 10 rh method of mixing. Sometimes this is vcry important, as the pl"l'S("I~(T of Oil' constituent is Il c ssary to pr vent others Iron: react.11 wu h cnch 01 her, or a reaction may ( . .ur which is dangerous.

(4) Some chemicals (e.g., sodium carbonate) may be supplied in the crystalline form, that is, having the natural water of crystallization; with this water all removed (anhydrous); or with a part of it removed. The actual strength of the chemical depends upon rh' amount of water present, so that one must know which is called for in the formulas and if that he is using is not of the strength specified, an adjustment must be made in the amount used so as to meet the spccifications of the formula. How important this is will be apparent when it is appreciated that crystal sodium carbonate is only 37% as strong as anhydrous. Unfortunately, the anhydrous absorbs water by standing, after being opened, to the extent that its strength may decrc'ls.c to 85% (i.e., the monohydrated form). The monohydrated salt IS stable, and hence is jhe most desirable form to stock. Should a formula call for anhydrous (desiccated) carbonate, the amount should be increased T 5% when the monohydrated is used. On the orh'l: hand, should a formula specify crystallized carbonate, the amount 0/ monohydrated used should be 43 less. If des~ccated (anhyd rO.lls.) carbonate has been stocked and exposed to the air for some time, It IS better to assume it to be not over 90 to 95% of full strength (i.c., which is about 98%) and increase the quantity called for according! . (5) Temperatures up to 125 F. are often recommended for mixin ' developer solutions; the primary purpose is to effec~ :ready solu tion of chemicals less soluble in cold water. When rmxing conccn trated stock solutions it is often necessary to utilize the higher tCIlIperature in order to get everything in solution. But it should never be exceeded and if time permits, it should be kept lower to be on the safe side. After mixing, a solution should be allowed to cool to normal room temperature before being used.

ro

DEVELOPING

SOLUTIO

IS

The following are especially recommended of negatives.


STANDAHD THREE-SOLUTION

for the development


(P-])

PYRO DEVELOPER

~;to'k Solution A Sodium bisulphite Pvro P;lt:1SSi\llll hromid Wattr, to mal (

For warm tones Aooirdupois . . . .


140
2

Metric
9.8
00.0

grams
ou n

'CS

10
p

gra ins Oil ne S

1.1
1.0

grallls gr:ll1lS rru illS lit er

PHOTOGRAPHIC

PROCESSES, MATERIALS,

AND EQUIPMENT

FORMULAS

Stack Solution B Water Sodium sulphite (desiccated) Stock Solution C Water Sodium carbonate

Auoirdupois . . 32 ounces 3"!-ounces 32 ounces 2t ounces

Metric 1.0 liter grams liter grams

La

days, if covered with a floating paraffin lid, the full size of the interior tank dimensions. For tray use take one ounce each, of A, B, C, to eight ounces of water and develop for 7 to 9 minutes at 65 F. For tanks use twice the amount of water and develop for 10 to 12 minutes.
METOL-HYDROQUINOKE BORAX DEVELOPER

(desiccated

)"......

75.0

For tray development use one part of each, A, B, C, with seven parts of water. Develop about 5 to 7 minutes at 65 F. For tank development use twice the quantity of water and extend time of development to about 12 minutes at 65 F. This is the time-honored standard pyro developer. By increasing the quantity of carbonate (Solution C) used, more contrast can be secured, and hence a considerable range is possible in the results obtained. The greatest objection to this developer is its rapid oxidation after being mixed in the dilute proportions employed, with consequent staining of the negatives, to say nothing of the staining of fingers. For tray development it should be mixed fresh from the stock solutions for each ncgative; also, for each batch in tank development, fresh solution should be used.
METOL-PYRO DEVELOPER

For medium Water (at about Metol Sodium sulphite Hydroquinone Borax Water, to make 125 F.) , (desiccated)

contrast, . . . . . .

(0-76) "with fine grain Avoirdupois 96 r 16 I3-} 290 116 ounces grains ounces grams grams gallon

Metric
3.0 8.0 400.0 20.0 8.0 4-0 liters grams grams grams grams liters

up to

(0-7) Metric 7.5 7.5 30.0 4.2 1.0 1.0 150.0 1.0 75.0 t liter grams grams grams grams Iiter liter grams liter grams

For warm Stock Solution A Water (at about 125 F.) Metal Sodium bisulphite Pyro Potassium bromide Cold water, to make up to Stock Solution B Water Sodium sulphite (desiccated) Stock Solution C Water Sodium carbonate

tones Avoirdupois . . . . . . . . . . 16 ounces ounce ounce ounce grams ounces ounces ounces

This developer is especially fine for panchromatic plates and though intended primarily for tank use, works well in tray development, where thc time can be spared for rocking the tray during the rather extended development period. Use the developer without dilution; for tank development 12 to 15 minutes at 65 F. is required. With tray development and agitation by rocking,7 to 10 minutes is satisfactory. .
METOL-HYDROQUlNONE DEVELOPER (0-72) on fast orthochrornatic plates

t t
60 32 32 5

For average contrast

(desiccated)

32 ounces 2t ounces

Stock Solution Water (at about 125 F.) Metal Sodium sulphite (desiccated) Hydroquinone .................... Sodium carbonate (desiccated) . .. . . . Potassium bromide. . . . . . . . . . . . . . . . . Water, to make up to . . . . . . . . . . . . . . or avcragc contrast

Avoirdupois
16 45 It 175 2* 27 32 ounces grams ounces grains ounces grams ounces

Metric
500 3.1 45.0 12.2 675 1.9 1.0 cc. grams grams grams grams grams litcr

use one pllrt of stock .solution to. on~ of w~tcr and

This modified pyro developer is a big improvement on the standard three-solution pyro developer so far as keeping qualities arc concerned. Several negatives can be developed in the same tray solution before discol oration becomes objectionable and tank. olutions can be kept for several

develop 4 to 5 minutes at 70 F. When maximum density IS required use the sro .k solution full strcngth. D-72 is a good all-around univ .rsnl dcv lop I' for pl.at 5, films, lantern slid's, and pap 'J', when used in Ihi: propl'l' t'Ol1ce11lrnUOIl for each. For

PHOTOGRAPHIC PROCESSES, MATERIALS,

AND EQUIPMENT

FORMULAS DEVELOPER FOR BROlVIIDE ENLARGING PAPERS (55-D)

paper prints with cold tones use diluted one to one, and for warmer tones, one of developer stock solution to two of water. .. For warm tones on lantern slides, Eastman recommend with their plates as follows: Soft Lantern plates. - I part stock solution to 4 of water; develop 2 to 3 minutes at 70 F. Medium Lantern plates. - I part stock solution to 2 parts of water; develop I to 2 minutes at 70 F. Contrast Lantern plates. - I part stock solution to I part of water; develop 3 to 5 minutes at 70 F.
CONTRAST DEVELOPER (DI I)

As recommended

by Defender

Co.

Stock Solution Avoirdupois Water. . .. .. .. .. .. .. .. . .. .. .. .. .. 32 ounces Metol 36 grams Sodium sulphite (desiccated) It ounces Hydroquinone .................... 144 grams Sodium carbonate (desiccated) . . . .. . ~ It ounces Potassium bromide * 60-144 grains

Metric
1.0 2.4 36.0 10.0 36.0 4-13.0 litcr grams grams grams grams grams

"' The liberal use of potassium bromide even in excess of the quantity recommended.

called for, is

For strong contrast

on plates and lantern slides

Avoirdupois
at about r z y F ) W_ater (b Metol . . . . . . . . . . . .. . . . . . . . . . . . . . . . . Sodium sulphite (desiccated) Hydroquinone .. . . . . . . . . . . . . . . . . . . . Sodium carbonate (desiccated) Potassium bromide Water, to make up to . . . . . . . . . . . . . . .

Metric
500.0 1.0 75.0 9.0 25.0 1.0 5.0 cc. grams grams grams grams liter grams

.
. .

16 14

2t
130 360 70 32

ounces grams ounces grams grams grams ounces

For use, take one part of stock solution and add two pans of water. Develop for from I t to 3 minutes. Short development makes for warm tones, the longer development for cold tones.
AMIDOL DEVELOPER

For prints, bromide

enlargements,

and lantern slides

Avoirdupois
Water.. . .. . .. .. .. ...... .. .. .. . .. .. .. Sodium sulphite (desiccated) . . . . . . . . . . rnidol Potassium bromide 16 45 15 ounces ounce grams grains

Metric
500 30 3 cc. grams grams gram

Use without dilution for either tank or tray development, when maximum contrast is desired. 3 to 4 minutes is usually ample in an agitated tray and 5 minutes in a tank. When less contrast is required dilute one to one.
DEVELOPERS FOR PAPER PRINTS

Although for paper D-72 can be used sati~factorily, better scale values will result from the following:
PRI TT DEVELOPER FOR WARM

warmer

tones and

Amidol developers must be mixed up just before using and should be used the same day. The proportions can vary over a considerable rangc for different degrees of contrast. Increase of the sulphite and bromide at the expense of the ami dol tends toward further softness; increase of the amidol percentage moves the effect in the other direction. FIXI

To 16 22

TES (D-52)

Stock Solution Water (at about 125 F.) ..... ...... Metol . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sodium sulphite (desiccated) Hydroquinone . ................... Sodium carbonate (desiccated) ... . . . Potassium bromide. . . . . . . . . . . . . . . . . Water, to make up to . ..

Avoirdupois
ounces grams ounce grams ounce grains ounces

Metric
500.0 1.5 22.5 . 63 15.0 I. 5 1.0 cc. grams grams gTams grams grams liter For fixation of lantern color tints. Hypo Water,

PLAIl

G SOLUTIONS Hypo SOLUTION


with water

slides which are to be hand colored

!
90

t
22 32

to make up to
I

64

pound ounces

For-use, dilute one to one and develop for about I! minutes at 70 F.

For acid hYI 0 to be used for general fixation, add to above from ~ to .\ Iht- <juanl irv of the following stock solution, dcp nding on the amount of h:lJ'(lming'desil'ed. In hot \\' m her I he SI I'ong T solution will h - required, IIIIt il 111:1)' not he necessary ill cold \\ t':1I IIn.

PHOTOGRAPHIC PROCESSES, MATERIALS,AND EQUIPME ACID HARDENERSTOCK SOLUTION (F-Ia)

FORMULAS Use for rinsing plates, films, and paper prints between the dcvclopin and fixing processes. It not only stops development but neutralizes ;1 part of the alkali of the developer, which would otherwise be carried over into the hypo solution and cause discoloration of the latter, through oxi dation. REDUCING SOLUTIO For Plates and Films JS and that
I

Avoirdupois
Water (at about 1250 F.) Sodium sulphite (desiccated) Acetic acid (28 %) Potassium alum. . . . . . . . . . . . . . . . . . . . . . Cold water, to make up to . . . . . . . . . . . . 56 8 24 8 ounces ounces ounces ounces gallon

Metric
1.7 240.0 750.0 240.0 4.0 liters grams cc. grams

liters

In case the acetic acid is stocked as the glacial acid, the 28% acid is made by diluting 3 parts of glacial with 8 parts of water. CHROME ALUM FIXING BATH (F-I6) Recommended for plates and films, in hot weather Solution A Hypo Sodium sulphite (desiccated) . . . . . . . . Water, to make up to . . . . . . . . . . . . . . Solution B Water Potassium chrome alum . . . . . . . . . . . . Sulphuric acid, concentrated ........

The most useful reducing solution for the photomicrographer which may serve all practical purposes for him is: FARMER'S REDUCER Stock Solution A One to two ounces of potassium ferricyanide Stock Solution B Ordinary plain hypo solution, in [,6 oz. water

Avoirdupois
2 2 3 pounds ounces quarts quart ounces ounce A rapidly.

Metric
960.0 60.0 3.0 1.0 60.0 8.0 grams grams Iiters liter grams cc.

16 oz. of hypo to 64 oz. water

Pour solution B into solution A while stirring As this bath loses its together in quantities films should be rinsed rinsing can take place

hardening properties rapidly, it should not be mixed greater than needed for immediate use. Plates and very thoroughly before placing in this bath. The in part in Bath SB-3 if latter is necessary.

CHROME ALUM HARDE ING BATH (SB-3) For Plates and Films Water Potassium chrome : alum . . . . . . . . . . . . . . . . .. I I quart ounce

This bath can be used in hot weather between the development and fixation, and with either F-I 6 or the regular acid hypo bath. It should be renewed frequently. SHORT-STOPRI SINGSOLUTION(SB-I) Water Acetic

d ounces (or t oz. glacial acid)


acid (28 %) r quart

This reducer is a surface cutting or subtractive one, and hence will ill crease the contrast as it reduces. T-he action is not strictly con f n d to this result, however, as by a change in the concentration it may become more of a proportionate reducer and not yield so much increase in con trast. The greatest contrast is obtained with a relatively strong solut ion of the ferricyanide. For use, about one ounce of hypo solution should be added to seven ounces of water and a little of Solution A added to this, just enough to give a yellow color to the hypo solution. This may be seen better if it is added in a white porcelain-lined tray. If the reduction is to be made on a wet negative, thorough "washing out of the acid hypo should be done first. If the negative is already 'washed and dried, it should be wet thoroughly in water before placing in the reducer. 1h ' tray should be rocked slightly and the negative examined frequently until the desired density is secured. It must then be rinsed under running' water for a few seconds to remove most of the reducer or reduction will continue beyond that desired. This is followed by washing in the usual way as done after the ordinary hypo bath. This solution does not keep for more than a few minutes after b ing Il .ixcd together, and hence if tine desired reduction is not sccur cl rlIrcr three or four minutes, a fresh bath should he used and the proc'ss continued until the desired rcdu tion has b en brought about. Tn most .nscs, how .vcr, when contrast), fT crs ar l' iquircd, the reduction should have hccn completed within onc minute.

PHOTOGRAPHIC

PROCESSES, MATERIALS, REDUCER

AND EQUIPMENT

FORl'1

LAS

<J7

PERMANGANATE

(Rs-a )

Stock Solution A Water Potassium permanganate Stock Solution B Water Sulphuric acid (concentrated)

. . . .

quart

I! ounces
quart ounce (Pour acid into water)

the following formula is included. It will be noticed that this formula i, a combination of the permanganate (subtractive) and persulphatc (~l~p 'I: proportional) reducers, since no one chemical is known which by Ir~ .lf will give a strictly proportional reduction.
PROPORTIO TAL REDUCER

(R-S) quart grains (03 gram) ounce (16 cc.) quarts ounces

For use, take 1 part of A, 2 parts of B, and 64 parts of water. When the negative has been sufficiently reduced, place it in plain or acid hypo for a few minutes to remove yellow stain, then wash thoroughly. The only advantage this reducer has over Farmer's is with plates that have been developed in pyro developer and stained excessively in it, since it aids in removing the brown developer stain. Also, by the use of a very minute quantity of the acid, Solution B, it can be used to remove" dichroic fog" from the surface of a negative, but if the acid be insufficient a staining of the negative from a precipitate of manganese dioxide results. This latter can be removed in a sodium bisulphate solution.

Stock Solution A Water Potassium permanganate Sulphuric acid (10% solution) Stock Solution B Water Ammonium persulphate

. .

t
. . 3 3

For use, take one part of A to three parts of B. Use same precallt~()ns as with other types of reducers. When reduction is s~cured, wa h 111 a solution of sodium bisulphite, then in water, before drying.

INTE
AMMONIUM PERSULPHATE REDUCER (R-I)

SIFIERS

Water Ammonium persulphate Sulphuric acid (concentrated)

. . .

3.0

quart ounces cc. (! dram)

For use, dilute with two parts of water. Ammonium persulphate is a super-proportional reducer, and hence reduces contrast as it reduces. This is due to the fact that it acts faster in proportion to the amount of silver present. It is important that the negative to be reduced be thoroughly washed before reduction, to assure uniform action, and the reduction must be watched carefully since the action ultimately attacks the shadow details and the negative will then be ruined. After reduction and superficial rinsing, the negative must be placed in a solution of sodium sulphite to stop the action and neutralize the persulphate remaining in the emulsion. It should then be washed in water, as usual. These two types of reducers should be ample for practically every condition that is likely to occur in photomicrography, since slight flatness or cS)!1trast can be corrected by the use of the corrective grade of printing paper. However, for the sake of anyone desiring a proportionate rcdu . .r,

Although, as pointed o~t in the text, intensification should not be re sorted to in photomicrographic work 'when it is at all possible to I.' tal<t' the negative, for an extraordinary occasion it ~ay b~ necessary to II1IC~l ify a negative, and hence at least one type of 1l1te.nslfiel~ shou~d be avail able for the purpose. There are many different I~~enslfiers JI1 use, cm ploying silver, mercury, and chromium as the add.lt1ve metals. Mos~ of these involve at least a two-step process, bleaching and redcvclopin r. Probably that which is the least complicated to employ and which results in a fair degree of intensification is the following:
MERCURIC-IODIDE INTE SIFLER

Mercuric iodide Potassium iodide Hypo Water

. . . .

2 2

z lOO

grams grams gra I11S


CC.

'ariv s should .bc well washed befor

placing in rh' solution.

Th
I ,

action is progr .ssivc and should he watch .d until the d 'sir .d d .nsit y .has been rea -hcd. III order 10 make the 11<.' rnt ivc permanent, :Ifl<.'r W:1SIIlIl

FORlvI LAS PHOTOGRAPHIC PROCESSES, MATERIALS, AND EQU[PME T

()!)

it should be immersed in a I % solution of sodium sulphide until the image, when viewed from the back, has been changed from a grayish to brownblack color. It is preferable to mix up the small quantity of this intensifier as required, or it can be purchased in prepared form, which is very convenient. MISCELLANEOUS The following miscellaneous formulas will be found convenient times, in connection with various phases of photomicrographic work.
CLEANTNG SOLUTION FOR TRAYS
I . quart

general retouching is not desired. For use, apply a drop to thc nrcn 10 Ill' corrected and rub over with a tuft of cotton, leaving only enough of t hc dry solution on the surface to receive the pencil marks.
DESENSITIZING SOLUTION

at

Water _ Potassium bichromate Sulphuric acid (concentrated)

. . .

3 5

ounces ounces (add slowly to the bichromate solution)

When it- is desired to watch the development of panchromatic plates under a fa-irly bright light, the plates may be desensitized by a preliminary soaking in a solution of a dye known as Pinakryptol Green. It is used in a strength of one part of the dye to 5000 parts of water. Two minut 's' immersion is sufficient. This part of the process mu t be carried on under the series 3 safelight, or in the case of super-panchromatic emulsions, in absolute darkness. After the treatment, and after the plates have I> .n placed in the developer, a fairly strong light, such as that employed for the development of process plates, can be used. On the whole, howcv '1', the photomicrographer will do better if he does not worry about th ' :1p pearance of the image during development, but relies entirely on the COil I bination of proper exposme, time, and temperature of development, .1 (I produce the correct result.
REMOVAL OF STAI 'S FROM THE HANDS

This solution should be kept on hand at all times for the purpose of rinsing out trays and removing the silver discoloration which occurs, especially in the developing tray. A tablespoonful of the solution is usually sufficient. After clearing the tray, it should be rubbed thoroughly with a wet cloth or tuft of cotton, then well rinsed in water.
FF.RROTYPE SOLUTTON

For treatment

of japanned

plates to prevent

sticking

Users of pyro developers may expect stained fingers if they are allowv.! to come in contact with the solution; the fingernails are the greatest ~uf Ierers. This stain can be fairly well removed by soaking the fingers in :\ 200/ solution of potassium permanganate. This oxidizes the stain but IT suits in what appears to be an even worse stain. This latter is quite casil) removed, however, by a second washing in sodium bisulphite solution or in a weak solution of oxalic acid. Frequently a little developer, not only pyro but M-Q as well, may 'l't on a good white shirt in the. darkroom an~ not .:be detected until a d:\1'I brown stain shows up some time later. This stain remover works equally well for removing such stains, which otherwise will remain in th goods after an indefinite number of washings.

Dissolve a piece of paraffin wax about the size of a small marble in six ounces of benzol or xylol. Keep well corked. Apply a little to the surface of a plate and rub over until the solvent has evaporated. Then polish thoroughly with a clean soft cloth until all smeariness has been removed. Repeat whenever there appears the least tendency for the plates to show sticking.
RETOUCHING SOLUTION

Dissolve about a teaspoonful of Canada balsam in four ounces of benzol. The exact concentration can be varied to suit the user's individual re quirernents. This solution is required rapher, to correct artifacts only on rare occasions by the phoromicro , in a specimen - dirt, torn tissues, ClC., sinc

ILLUSTRATIVE PHOTOMICROGRAPHS

301

, Chapter ILLUSTRATIVE

PHOTOMICROGRAPHS

Among th~ best methods for improving one's technique in any field of endeavo~' IS tl:e stu,dy of the work of ?thers, Photomicrography dO,es,not dIffer m this respect from ordinary photography, music, pamtl,ng, or other arts where personal ability plays a part, The underlymg theory back of this practice can be stated in the old-time proverb, "What one fool has done, another can do," :t:Jaturally some lines of work require an inborn genius, without which all the practice and experience that micht be crowded into a lifetime would be useless, to , In others, latent tale1:t plays but a small part; continued experience IS~lore largely responsible for final results, Photography and, photo~lcrography ~re a1!10ngthese latter. This is because, in the last analySIS,~he work I~ 1ilamly the product of mechanical (including optical) devices, c?~nbmed wltl~ natural or producible objects, and not all of human ability. For tl11Sreason, havinz before one evidence of what is possible provides for the novice, as it ~vere, a mark to shoot at. It is for this rea~on that representative photo micrographs depicting many types, of objects and numerous variations in technique are included m this chapter. These have all, without exception, been chosen from among the many thousands taken by the author, for one purpose or another, during his past forty years in the work. ~ot all those show11:are perfec.t in eve~y respect, nor do they necessanly represent especially beautiful subjects such as one rnicht pick out for ~ competiti:re exh~bit of photomicrographs. Rather, ~11anyof the~n will se~m qmte ordinary to specialists in the particular field in which ,the mIcrographs would be classified. In only a few instanc S have pI~ture? been included which possess general interest; the main emphasis ha~ been placed on illustrative examples covering a l:lrgc number of lines ~f w?rk. That the selection of the pictures shown has been made with difficulty may be appreciated when it is rcaliz .d 300

that, in the realm of Haematology alone, the author has taken upwar?s of a th?usand photomicrographs, while in Metallurgy the nUIlI bel' ISmany times this figure. Yet these two subjects are represent 'd in the list by a paltry half-dozen. . There will be found examples of low-power work, by both trans mitred and incident light, and also of extremely high magnifications, in the region where, according to all theory, " empty magnification" should run riot. Examples of optical sectioning and great depth of focus are also shown. Tl~ere are many fields of specialized microscopy not represented in the list. These include such subjects as fluorescence microscopy, chemical microscopy with polarized light, colloidal microscopy, g 'neral ultra-violet work, Rheinberg illumination, powdered materials (pig~ents, activated carbons, fillers, powdered talc, etc.) , dis 'as' fungi, commercial fibers, and others. Lack of space was responsihl ' for their elimination. An accurate check of every photomicrograph taken in the counrry today would probably show a preponderance of them to fall in t 11 ' realm of pathology; it may seem, therefore, that this field is not pro portionately represented in the list. It was felt, however, that the subjects themselves, being so highly specialized, and understandable only to the medical profession, and of so little interest to the avcra r ' rnicroscopist, did not justify the placing of emphasis upon this type of photomicrograph. The technique involved in this subject does not differ from that called for by histology and other biological sections; therefore, these latter can serve as a guide to the pathological photomicrographic technician. It is hoped that the method of presenting the pictures with an indi vidual explanation of the subject, special technique, and general prob lems involved, together with the photographic data, will increas t hci I' value as practical examples to workers in various fields and at the same time make them of general interest to those not especially on met! with the specific fields represented by some of them. Some of the pictures may be of interest to biologists even though they may not I> interested in photomicrography, because of the unusual features ill rh objects themselves. I-lad space permitted, this phase of the subjet't would have received more attention, as some very uniqu biolo ricnl phororni rographs could have been shown, rep res nting rare and lit t 1,1 nown condirions. In the t .chni .al data on the pi .turcs, it will be noted that in l'CIH.'I':tI

302

ILLUSTRATIVE

PHOTOMICROGRAPHS

the camera extension has not been given. The primary reason for this is that the figures on the record cards would require re computing to mean anything to those not using the same type of equipment. The graduation in centirneters on the camera extension bar (the figure recorded) is not necessarily the distance of the plate from the eyepiece. In other words, the distance of the eyepiece from zero would also have to be stated, and this record has not been kept. It should suffice to know the lens combination employed, and the magnification. The magnification specified can be secured only at one particular camera length, should anyone desire to duplicate the conditions. If magnification charts are made for each lens combination, as suggested in Chapter 4, it will be possible for anyone immediately to set his own apparatus at the proper camera length, to reproduce the results. Where it is of importance, to emphasize the fact, mention is made of the part played by employing an unusually long projection distance. The exposure time cannot always be used as a guide for duplicating results on other equipment, inasmuch as the nature of the light source, the density and stain of the section, or other peculiarities of the object, are not necessarily comprehended from the photograph, and may vary over a wide range under two different sets of conditions. It will be apparent, from study of the exposure data, that the light source most commonly employed was a 500-watt lamp. This was seldom used direct, as in the so-called Kohler illumination, but as in the author's method described in Chapter 4 (page 192), with two pieces of frosted glass directly in front of the light. The transmission curve of these pieces of glass is shown in Figure 12 I, indicating substantially 50 per cent reduction in illumination. This fact must be taken into consideration in making comparisons of light intensities. It has not been possible in all cases to give the exact stain used on the section or specimen but an attempt has been made to give an idea of the general color shown by the slide. Data regarding the prints have not been included. With very few exceptions, paper of #2 Grade (Normal) of hardness has been used, always in glossy finish. None of the pictures shown are enlargements; in every case they are contact prints from the negatives. It is probably needless to add that some of the finer detail which is clearly visible in the originals must of necessity be lost in a half-tone reproduction.

PLATE

1.

STEM OF CANE, TRANSVERSE SECTIO MAGNIFICATION 8Sx

Medium-power views of botanical subjects, such as sections of plant stems, ofT'r a fine starting point for beginners in biological photomicrography. They are lIs~I:tII)' contrasty and more easily interpreted than animal tissues, because of the conrinuul repetition of the structural elements. With respect to the spectacular aspect, of tell :111 asset in the first pictures taken, endogenous stems, such as that shown here, arc su perior to the exogenous. Sharp delineation of the structures - cell walls, etc. - will be one indication of :t successful micrograph. Exposure Objc iiv F)' 'picce Condcnsvr
16 III III ,

Data Illuminat ion 50()-watt I:ull]! Plnt e \VrnuclI 1\1


Filtl'r
F\I)()~III't'

(IOX)

apochrornat

110011al 11 , '4 . . alllalll1t


I"<'t

B (green)
\', \(,(,1)))(1

StTtiol1 Maim'.! light

PLATE

n.

VASCULAR CYLINDER IN ROOT OF RANUNCULUS, TRANSVE.RSE. SECTION MAGNIFICATION 300X

PLATE

HI.

PROTOPLASMIC THREADS IN SEED OF DIOSPYROS MAG IFICATION 750X

. W~len success has .been achieved with low-power pictures, an increase in magnificanon IS the next logIcal step. A single fibrovascular bundle will serve as a suitable subject. It should be enlarged to the point where each type of cell can be differentiated easily from the others. This micrograph, showing the central vascular cylinder in the root of the common buttercup, gives a good idea of how a satisfactory view at a medium high power should appear. The contrast between the clear background and the various COIlstiruents of the section should .be sufficient to eliminate a muddy or foggy appearancc, yet there should be ample difference III the cells so they do not all appcar a uni form black.

The photogl"aphy of a botanical section su~h as shown in this micrograph rcprcsenls a further advance III the technical problems involved. Not only IS the magnification considerably higher - made necessary by the fineness of the protoplasmic threads between the cells, which constitute the particulul". item of interest in the subject - hili careful attention .must be glVen to securIng a semi-optical section. Thc seed of DIOSPYWS, the Japanese persimmon, is composed of sclerotic c lis (i.e., like a date seed), but passing through the bonelike structure of thc cell walls arc 1ll1111~rOLlS capillaries, serving to connect the protoplasm of the adjacent 'ells aud III ovidc COI1lIllLlnlcat~on between them. Special staining techniC],uc is nccessary to make these thrca Is VI .iblc. They arc analogoos to the canaliculi III bone (Plate 10),

Exposure
Objective Eyepiece Section 16 mm. (IOX) - Homal I stained with apochromat

Data Illumination soo-wan 1:1I111' Plate - Wrnncn M Filrer C (0I'1\11~'t) 1':XjlllHlIl'I: :5 S ('''I His Objective

Rxposnre H 111111, (wx) apo 'hI'OI\1i1L


I':)'l'pil'('~ 11<>1\\:11 I (;olldl'IISl'1' '''1 N.i\. 11]111111111 S ('lillll HI:till\\d wII" irnn 11I'"I,III")'1i1

Data
1I111111il1:lt'i0I1 5()O-Wi1(t 1:1111]1 PI:\I\' \ Vnu 1\'11I'd Filll'I' (; (1II'lllllo((') I' I 111'1' I ~ '(1('01111

Condenser - [.4 N.A. aplanat


hcmatoxylin

and snfrnnir:

'I"

PLATE PLATE

V,

GLOEOCAPSA G STATE

IV,

SPIROGYRA
230X

SHOWN

IN THE LIVI

MAGNIFICATION

MAGNIFIED

2000X

In spirogyra, the sl!my "fog, spittle" of ponds, and ditches, the microscopist finds one of the most beautIf~ of objects, The cylindrical filaments with their green spiral coils, ~he spaced pyrenoids and th~ centrally placed nucleus, with its radiating protoplasml~ threads, o~er the photomicrographer a fine opportunity to test out his skill. A pIcture of this alga, to be successful, should reveal all the structural details in such a manner that a natural appearance results, Exposure Objective 16 mm, (IOX) apochromat Eyepiece - Homal I Condenser - 1.4 N.A, aplanat Specimen stained with methyl violet Data Illumination - 50o-watt lamp Plate - Wratten M Filter - B (green) Exposure - 25 seconds

Gloeocapsa is a minute unicellular alga, somewhat colonial in habit because of rh ' presence of a thick gelatinous capsule surrounding the cells, This holds them together 111 groups of two, four, etc. The cell itself contains chlorophyll, and hence is grc '11 in calor. Thc specimen shown here was photographed in the living state, unstained, in wnt cr. Whcn four individuals occur together, as is the common condition, there arc rhrcc sl'paratc capsules present, A good micrograph should show these, It is easily ac xnuplishcd by stopping clown the cone of illumination with the subsragc cliaphr~gl1l, This is onc typc of photomicrography that requires thc use of the microscope ill u V .rricnl position,

Exposure Data
Ohject ive Zciss X npo .hromru (3111111, ,H5 ,A ,) F)'l'pil'l'l' l lomul III (:olldl'II~I'I' I", "llplnl1l1t, I't'lhlt'l,d ,lpt'l't 111'1' Sp\'\'IIIU'1l lil ill~, ruuuntvrl III rrt'~h 1I'llt'" llluminntion 50U-wlltt hltltp Plm e \ Vrnuen M Filll'r (; (fl'llll~t'l I, 'IHl 111'1' 1111111111\'\

PLATE PLATE

VII.

DIATOM, SURIRELLA GEMMA

VI.

ANIPHlPLEURA PELLUCIDA

lVIAGNIF1CATION

6000x

MAGNIFICAT10N

4500X

Amphipleura pellucida has long been the classic test for high-power objectives and for determining the skill of the rnicroscopist, To be able to resolve the markings into transverse lines is a feat requiring considerable ability, while to show the dots represents the utmost in microscopy, both on the part of the lens and its possessor. At least, so thought the old-time microscopists, The fact that specimens from different localities vary greatly in the ease with which they can be resolved into dots has been generally recognized. This difference materially discounts the results achieved in any case, either in visual or photomicrographic work. Of all the slides of this diatom in the author's collection, mounted in all sorts of media, some cannot be resolved into dots at all. The best specimen for dot resolution happens to be mounted in balsam, and is therefore hard to photograph so as to yield good contrast. The specimen shown here, at a direct magnification of 450ox, is mounted in realgar, but is only mediocre so far as dot resolution is concerned. Nevertheless the dots can be seen in the picture. The use of light of shorter wnv ' lcngeh increases the resolution but this cannot be done with realgar mounts because rcnlgnr is a str~ng yellow filrcr and cuts off all ultra-violet light.

, The photography of diatoms probably offers a greater temptation to go the limir III the matter of empty magnification, than any other field of microscopy. To obtain resolution of the marktngs on some of the finer-marked species is, in itself, a cause for g~atlfic:tlOn. Naturally, therefore, to reveal these markings plainly in a higllpower micrograph IS a further goal toward which to strive. Surirclla gemma is not a difficult diatom to resolve for the markings run around ('o,oo? to the inch. Neither is it a difficult diatom to photograph, although the surface IS very uneven. But to obtain a. direct magnification of 6ooox, without an ob )cctlon:lb.l~ al~lount of fuzziness, reqUIres the best quality of obj~ctive and a good phOlOIIlIC[()!?aphlc outfit. ThIS micrograph IS included here to illustrate what 'HIl be accomplished, should one's inclination lie toward the realm of super-power IllHglllfieallons,

Exposure Oh] ct ivc


2

Data Illumination 5 a1l1p Plnll' \VI':III'II M Ililll'!' C (blue-violet) F\llIlhlll'l' I Iltilllll('


1"

II1IIl. ',4 N,/\. npo{'hro-

Expostrru Data
Objective 111:11 111'pil'I'I'
2

111:11 11~)'l'pill(t' Zt'iss # r z l'<III'Ill'IISlIlillf.( ( :011 tit, 11~1'I' \VI1I~OIl l lolos, oillltl, 11 illl liglu

HI"

111111. 1,4 N./\, :qlll('lIl'lI-

1111111011 1\'

1I111111illlll ill 11 III 111111"'1'1' .11'1' 1'1.111' I 1111111 I 11' I I I III I 11 11'1 ( (111111\ 11111' I)

PLATE

IX,

SLIPPERY ELM

WOOl)

(ULNA

FULVA) , TRAl

VERSE-SECTlO

MAGNIPIED 40X

PLATE

VIII,

LEAF BUD OF HICKORY

iVIAGNlf"ICATIO r 4X

An opaque object such as this offers the photomicrographer an opportunity ~o meet unusual conditions in low-power work, In the first place, the ,stem, IS cylindrical and of considerable diameter, so that the depth of focus req ul!:ed IS excessive. The next problem is to illuminate the stem so that no part \\,I~l be ..'~1d ep shadow, and at the same time the terminal buds must show suitable relief. I he ha 'kgrolll1d must be such that it will not detract from the object, Diffuse ligh, in,g, n IO!\f{-(o 'us objective, working at a small, aperture, and a COl'!''Cl' .c~posllr', provide Ihe :lIl~wcr, The background is a large whir cnrdhonrd, plnccd ~lInlt'lt'nlly (Ill' ,IIW!\~'111111 110shad o ws can (all upon it, Ihollgh il 11111'1 he nuiply illuminuu-d hy IIH' 11~11I~01I1('(', Ohjl'I'liI,' 11111111111, 1111111111' 11111:1(. I~IIII" \\'1'1111('11M /0.'1/1("/1/'1' 1l.,I,/ 1I111111il1lllillll 11111' I"" \1,"11:11111' IIl1d 1'liI"1 III1IH'
I

For identification purposes the transverse section of wood is the preferable on' to photograph, It requires three sections, however, to reveal the complete structure, a radial and tangential as well as transverse, Low magnification, such as this, will depict the annual rings, structural character istics, etc. better than a higher power. For study of the individual cells it is nee's sal')' to use a higher magnification, The photomicrography of wood sections usually is not different from that ern ploycd for other botanical sections, Some sections may be unstained and the necvs sHry contrast can be secured only by the use of non-color-scnsitizcd plate :1I1d :I reduction in the illumination cone, There is frequently a yellow tint ill Ihi' lignin which photographs best on plates sensitive to blue rays only At other lilllt'S woods ,11'e deeply colorcd brown or reel and r quire a rcd-scnsitiv and possibly 11 rt'd (ill cr. ":,"/10.1'111'1' Dnt a Ohjcrt iv 1O 1/1111. Plnnnr ]>1:11(' \\11':111('11 1\1 l lluminnt ion iOO 1111111111111' ]i ill ('I' Il (I 1'('('11) (:1111(/1'11'0"1' "'Ill, SI"'I'IIIl'II' 1"11'0, 11 I' '1111'0111'1' '",('olld'o 1111 I 111111'1 111\\ 1111\\ 1'1 1I11'llilld SI,IIIIII '01,11111,11 11lib 'o,ill,"dll

312

PLATE

X.

TRANSVERSE SEcrION MAGNIFICATIO

OF HUMA
300X

TIBIA

PLATE

XI.

COCHI.EA OF GUINEA

PrG, SHOWN

IN

Lot

GITUDINAL SECTIO

MAGNIFICATION

25x

The most interesting feature in bone structure is the presence of the fine canaliculi which radiate out into the bone from the minute lacunae. These are the channels by means of which the fluid communication is established between the various cells. A good microscopical preparation is necessary in order to re."eal them properly. Then the photomicrograph, to be of any value, must be of sufficient magnification to separate the individual processes. . . For a beautiful analogy to bone structure, III the vegetable kingdom, see Plate Ill, where the protoplasmic threads in a hard seed are not only quite similar in structure, but perform the same function. Exposure Data

In intricacy of design the auditory organ is a close second to the eye. That part of the ear where sound vibrations are transformed to nerve sensations resembles :I minute convoluted sea-shell. This micrograph gives a good idea of the .gclI~I':" form of this intricate apparatus, the cochlea, as it appears in a median Jonglludllllll section. The magnification is not sufficient to reveal the complexities of design bur it is that best suited to convey an idea of the .stfllcture as a whole. This type of P"()~O Illicrograph, covering a UI1lt structure 111 Its entirety, fills an Important niche III hlu logical photomicrography. Exposure Data Obj ici ivc 20111111. Plnnnr at Ji:()'3 llluminntion son-watt lalllp <:llIl(ll-IIS1'I' Zl'iss # 17 'I'l'l't ,,('!t- 11'11\ S\II,tioll slaill d with hcuuun \ IIIl IIIId I-IISill Filler Plan:

Objective - 16 mm. (rox) apochrornat Plate - Wratten M Eyepiece - Homal I ~ilter - B (green) Condenser - 1.4 .A. aplanat Exposure - 40 seconds . Illumination - 500-w3tt lamp Section ground, silver illlprcgllatt:d and count ,t'st:tillcd red

13 (gl" .n) \Vl'allell 1\1 '+ s tolHb

Exposur

314

PLATE

XII.

SPIRAL SWEAT DUCTS I' rvIAG IFICATIO


40X

SKIN PLATE

A'lTI.

33-HoUR MAG

CI'HClC EMBRYO, 11'ICATION 7SX

WHOLE

Mou

Thi~ is a type of subject requiring special technique to bring Out the presence of the spiral sweat ducts which pass through the thick outer portion of the skin on the palm of the hand and sole of the foot. The possibility of showing them as in this 111lcrog.raph anses from the fact that the walls of the ducts possess a slightly hicber refractive index than the surrounding tissue. On account of their spiral nature ~hey have all appreciable depth, so that a low-power lens must be used to get them 111 focus. .Then, by f~rther. reducing the aperture of the illuminating cone, the difference In the refraction bnngs.them prominently into view. The section IS purposely cut thick, to Include the complete spiral and hence, with reduced aperture, the cells of the rest of the tissues cannot be well shown at the same time. The depressions on the surface of the skin, constituting the " finger-print" structure, are evident, They are partly filled WIth dirt. Exposure Data Objective - 20 mm. Planar Conde.,nser - 2 cm. spectacle lens Koblcr Illumination with reduced aperture cction stained carmine Plate - Wrat ten Panchromnt ic Filter (, (orallgl'l 1':~p()s\1J"(; I I xccund

In the field of embryology the photomicrographer can find abundant material suitable to his needs. A series of stages in the development of a chick embryo, fron I thl" primitive streak to the fully formed body, provides pictures of the highest cducmionnl value for teaching purposes. In the stage shown in this micrograph'the early develop ment of the fore-, middle-, and hirrd-brain , the thickness of the neural tissue, t hl' still open anterior cleft between the neural folds; the formation of the prilll:1ry optic lobes; and the early condition of the ectoderrn and mesoderm, can all hl' demonstrated. The technique involved in photographing an entire series of stages is quire vuricd and extensive, the complexity of the subject increasing all the way from the pl'illli t ivc streak to the age where whole mounts can no longer be consid red in a mirr scopi .al classificaclon. I\XjJOSllfC Dnt
Ohj('("I ive
I \"('pi,"'" (,X

npOl"hl'()Il\;\t ' \, ''I,LUIIII, \I 11 h

lllumimu ion
!'Iiltl' \\'r,ltl,'11

50tl wnu 1:1111p

I 111111:11 11

(:olld"Il'l'r
tllp ,'t.llIl,

I"

1"\'11111\1'.1 1,11111 lit 11111111 \ 1111

I'IIH'I' 11 (rj"I'1I) I'hl' C (III,llll'I') I' I'll IIIl t, 'I" IIl1d'.

PLATE PLATE

XV.
MAGKIFICATlOI

SECTIO ' THROUGH

E rnu:

XIV.

EYE OF 30 MM.

RAT EMBRYO,
SOX

M.EDIAN SECTION

lVIAGNIFICATIQI

650X

In the entire realm of life, the eye is paramount in its function, marvels of structure, and multiplicity of unusual histological elements. Satisfactory photomicrographs of the eye depend in part upon the quality of the microscopical preparation. The picture shown here owes a large measure of its educational value to the perfection of the section. The photomicrographic technique itself is not Out of the ordinary and the subject as a whole is illustrative of average histological material: Its c?ief contribution to this series lies in emphasizing the advantagc of good microscopical preparatlons, when they are to be photographed. Poor sections cannot be expected to result in good pictures. The stain of this section was light, but compensated for by the use of a contrast filter.
EXpOSU1'e

About the most pnmltlve neural tissue known .is the nerve plexus (or network of nerve cells) occurring between the body cells of the Hydra. t:here arc s ~ler~1 different types of cells present in the two-layered body sac, each .with Its own II1dlvidual function. The nerve plexus serves to connect all these together so that th ' Hydra body as' a whole functions as an individual organism. The photomicrographic problem in this case, with the help of staining n~ctho,!s which differentiate the nerve plexus, is to penetrate the body layers of an enure animal (four layers deep, counting the two sides) and isolate the plexus so that it will :11'rear as such in the micrograph, without being complicated by an excess of other 'clls, On the whole, this is an extremely difficult picture to produce.

Data Illumination - soD-watt lamp Plate - Wrattcn IV! Filter - B (green) Exposure 11 S '('ollds
11\111" ,85 F,\'l'pil'('l' C;Cllldl'lI~l'I' (llljl'\'I \IIIIIIl'd

Exposure Data
Oh] .ctivc - Z iss X npochromat
. .)

Objective - 5X apochrornar Eyepiece - Homal U Condenser - 1.4 .A. aplanat, top lens removed Section stained with carmine

(3

with

l lonml
1 1 pllll'

11
uplnrun
'11

.H~

. \,

Illuminarion 500-W:1tt lamp Pint' Wrntt cn M Jiiltl'I' C (ornllgd F\pmlll'c S Sl'('Ullds

111111' ,1I1d 1nl

318

PLATE

XVII.

OCELLI ANI) COMPOU 'D EYES OF DllAGONFLY MAGNIFICATION I5X

Fortunate indeed is the microscopist when he obtains an occasional section whicl: is ideally diagrammatic. The section through the head of a dragonfly shown here fnll, into this class .. It is not only median through the ornatidia of the compound cyes bill also median through the two horizontally placed ocelli. Sections of this size require a low-power photographic lens to cover them. In this particular section the staining was quite deep, resulting in a contrasty negative, Exposure Data Objective - 50 mm. Planar 5 .m. condenser using Kohlcr rnctho I lllumination yoo-watr lamp S(:rlion stain d with hcmntoxylin Filter - E (light red) plus , (ornngc) Plate - Wrattcn M Exposure - 11 seconds

320

321

I
~,

PLATE

XVIII.

PLATE MouTH PARTS OF TSETSE FLY MAGNIFICATlON 30X

XL,,{.

THE EYE LENS OF A BODY LOUSE, SHOWN IN OPTlCAL SECTlON MAGNIFICATION IOOOX

In this object we encounter one of the conditions so prevalent in the photographing of insect preparations - the great difference in the intensity of color in various portions of the chitin. The specimen shown here was mounted by the pressure method, which involves a preliminary soaking in hydroxide to soften and remove internal tissues. This results in the head (a portion of which is seen in the picture) showing beautifully clear, but the hairs and other mouth pans are still a very dark brown. As in this case, however, we are particularly interested in the swordlike piercing beak, everything else must be subjugated to this pan in taking the picture. In other words, the dark portions must be allowed to show black. Should an attempt be made to lighten them (which can be done), the beak would become practically invisible. Exposure Data Filter - G (orange) Plate - Wratten M Exposure - J second

Though most insects possess compound eyes (as shown in section in prate XV 11), the" cootie," as it is familiarly called, has individual eyes, one on each side of the head. In a mounted specimen the lens stands vertically, and so provides an opportunity [or observing the surface contours. To photograph it, however, is quite a problem. lr reguires an optical section: and considerable magnification. The keratin of the exoskeleton is pale yellow in col or, so that, contrary to the usual condition when photo!l"raphing insects, increased contrast, instead of reduced, must be secured. The lells J tscl f is tra nsparent. The micrograph is interesting in the information it yields as to the difference in curvature of the outer and inner lens surfaces. It also reveals the presence of :I thin cuticle over the lens and exoskeleton, and that the latter extends slightly within the lens, possibly serving as a diaphragm to cut out the marginal rays. Exposure Dnt
~(l()

Objective - 20 Planar, slightly stopped Illumination - soo-watt lamp Condenser - Zeiss # 17 spectacle lens

Ohj('rliv(' H 111111, (20:,) npnchronuu F\'('pi('('(' I IOIlHJI COlld('lls('r 1,,1Ilplall:lt Ilflllllilllltioll \\,llIt ""lIp 1'1111(' 11111111111'1' Slo\\ FiliI'!' Sl'hlltl 11. light (lIght hi lid I\L1KIllfinltloLl11111111111'.1 1I11'llIlghIINI'III 1111lf( 1111111\\.. I' 'IIIINIIII' 71 ~I'I'IIII1I~

3 I

PLATE

XX.

MITOSIS IN SPERMARY OF POTOMOBlUS 1VIAG,IFICAT[ON 700X

PLATE

XXI.

MITOSIS 'IN SPERMARY OF POTOMOBlUS, POLAR VIEW OF CHROMO OMES iVIAGNIFICATION I600x

A general view photomicrograph should reveal as much information regarding the subject depicted as can be crowded into it. In one showing cell division, as instanced here, careful examination of an entire section will often disclose some particular area where an abundance of stages is present. The chromosomes are very numerous in Potomobius and are nearly spherical, and hence cannot be separated in an equatorial view. On the other hand, the manner of separation of the two sets of chromosomes, and their reforming to produce the daughter cells, is easily seen. This micrograph is especially interesting for its depicting of metaphase, anaphase, and telophase stages.

For a study of the individual chromosomes of Potornobius a polar view is ncccs sary. Also it is desirable that the magnification be considerably higher than ISrequired for a general view. In a photomicrograph of the chromosomes a compromise should be made in the matter of a resolution versus depth of focus. A magnification .of Ill.o()X requires fairly high aperture. in the objective in order to aVOId empty rnagriificatiou, but, on the. oth'er hand, if a critically sharp focal plane, without appreciable d 'pl 11, results, very few of the numerous chrom.osomes are 111focus on any o~le plane. The photography itself is relatively SImple because the stall1111gWith iron hCIlI:]toxylin gives a very dark color to the chromosomes regardless of the filter employ l,

Exposure Data
Objective - 8 mm. (zox) apochrornat Eyepiece - Hornal I Condenser - 1.4 aplanat Sectjon stained with iron hematoxylin Illumination - yoo-watt lamp Plate - Wratten M Filter - Schott I mm, 1I (light blue) xp05ure - 20 seconds

Exposure Data
npochromat (3 Obje tivc - Zciss 11\111., ,H5 ,A.) Fyt'pit'('" 1101\\:11III (;Cll\d"I\\"I' 1,4 uplnuut Sl'Clinn ".Iim'd \\ il 11iroll 111'1\\\1111 llluminntion 50o-wntt lamp 1'1:11,' Wrnucn M Fil\l'l' Schot\ I 111111. II (Ji!fhl hluv) I' pCl~III'" \0 ~" 'OIld\

324

PLATE PLATE

XXIII.

MATURATION

XXII.

MITOSIS IN HYACINTH ROOT MAGNIFICATlO

TIP, 1600x

LATE A

APHASE STAGES

IN ASCARIS EGG, SHOWING THE MALE AND FE PRONUCLEI lvlAGNIE'ICATlO


1200X

fALE

.Proba~ly the most interesting, as well as the most spectacular, stage in mitotic cell division IS the late anaphase. At this time the chromosomes are nicely spaced into the daughter cell groups, yet retain their individuality. Visually it is sometimes possible to follow through the course of single chromosomes in this stage, especial! y if a stereoscopic binocular eyepiece is employed, but this cannot be done in a photomicrograph, for two reasons. First, it is impossible to alter the focus while taking a picture, as can be done for visual obser:ration; and second, all chromosomes being stained dark, their Images superImpose into one general black area. However, in a good micrograph, a compromise focus should suggest the actual structure. Exposure Data Eyepiece - Hornal Hl Illumination - 500-watt lamp Plate - Wrarrcn Pal1(;hrolll:!lic Fxposur 15 ~l'l'<ll1ds

This micrograph reveals an interesting stage in the fertilization of an Ascaris egg . The male sperm has penetrated to the cel:ter of the egg cell but has not yet united With the female nucleus. The actual union does not take place until the haploid number of female chromosomes has been established. .In. the picture, the three polar bodies which have resulted from previous nu 1':11' diVISIOns are seen at the top of the cell, :vhere degeneration is taking place. 111 the cen.tel of the cell are t~1etwo nuclei which are to unite. It is impossible to Slate \Vh'~h IS male and which IS female .. Fusion is just about to take place. MI~rographs of this type, at a fairly high magnification, are of great value Ior leaching purposes. Exposure Datn Obj "live Zeiss X npo 'hI'OI11:1l ., ()o.\') (immersion, ,R) F)'I'Jlit,((, IlolI;:!1 III (;(JIH!\onM'I' 1..1 ' \, i1J1LiIIlII SI'!'II!>11 ~lililH'd \I idl il Oil hl'llIlll!> \Ihi 1!!ulllination SOO-wntt Fihrr C (ol':ll1{:(d Pill\(' \VI':lI\('1I 1 F'IH1'III'(' I IlIilHIlI'
lump

-'

Objective - Zeiss X apochromat (immersion, .85 .A., 60x) Condenser - 1.4 T.. aplanat Filter - E (light red) . Section stained with iron hcmnroxylin

3 7

..

PLATE XXIV.

CHROMOSOMES FROM SALIVARY GLAND OF DROSOPHILA, SHOWING THE GENES LOCI l\IIAGNIFICATION IOOOX

PLATE XXV.

GASTRULA STAGE OF SEA URCHIN, SHOWN IN OPTICAL SECTION MAGNIFICATION 600x

Research work on this classical object has been the means of materially advancing our knowledge of the factors infiuencino heredity, inheritance, mutations, etc., during recent years. The chr?moson:es of cl\e f~uit fiy are only four in number and are unusually large, hence easily studied. By SUItable prepal'auon methods It IS possible to reveal the component pans of chromosomes from the salivary gland, These show in the picture as alternate light and dark bands transverse th~ chromosomes. Extensive research has Identified many, at least, of the bands WIth distinct inherited characteristics, The chief problem in photographing these chromosomes lies in securing sufficient contrast between the rather faintly stained bands.

Exposure
Objective - 4 mm. (40X) B. & L. mersion fluorite Eyepiece - Hornal IV Condenser - 1.4 N.A. aplanat Specimen stained faint red

Data
Filter - B plus H (light) Exposure - [8 seconds Plate - Wrattcn Pan .hromnri Illumination - 500-W:1tl huup

In the development of the sea urchin embryo from the single-celled egg, the first division produces two cells, and these divide into four. Increasing in gcomcrric ratio, they become 8, 16, 32, 64, and so on. When the number of cells becomes .quire numerous, they form a hollow ball, the blastula stage. From the more numerous cells at one place on the sphere, a hollow tube of cells starts 'growing internally through the center, toward the opposite side. This is known as the gastrula stagc. 111 a stained specimen, all cells of the sphere, as well as those of the inner tube, arc stained alike; nevertheless, by optical sectioning, as described in the text, it is possible to photograph the inner tube and a ring of cells either above or below. Thcr is, of course, some interference, but the picture is easily interpreted. Especially lO Irv n .tcd arc the loose cells at the end of the tube, in the process of uniting the late 'I' to rhc opposite side of the sphere, after which the hole in the tube will be opened up :111the way through. The inner tube wall is plainly seen,
F:X/J().I(l'r .

im-

On/a
PIIIII' \VI'IIII'II

Ohjcci ivc .
E)'('pi 1('(' 1'1' (;Olltit'II ()Jljlll'l

111111.

(20.~)

IlP(1('IiI'ClIIHII

#H

M
i"O IVIII I

(,OIIlP('IIS11lilll' (fl'i~,)

llluiuinu:
Filll'l

ion
(; I

lulllpltllltll " dlll'p

111.1.
pllllt.

N. \,

lump

(01'1111111') ~11('lIll1b

~llrilll'd

I' P"

'"'1'

PLATEXXVI.

BACILLUS PARATYPHOSUS, SHOWINGTHE FLAGELLA MAGNIFICATION 2000X

PLATE XXVII.

The average pictures of bacteria one sees in various textbooks leave much to be desired. In the first place, the magnifications are usually insufficient to reveal the organisms properly; further, the photomicrographic technique is far from Ideal. With the majority of species of bacteria a magnification around 2000X IS desuab.le in order to convey a true conception of their form. Some need more than this, because of their' extremely minute size, while on the other hand a few are so large that magnification under iooo must be used. . . When bacteria are shown in situ in tissue sections, the latter is an additional factor governing the magnification, which usually cannot be so high. Unless typhoid bacteria are properly fixed and stained they do not reveal the presence of the flagella, seen in this micrograph. Exposure Objective - 2 mm. 1.4 N.A. apochromat Eyepiece - Homal IV Condenser - Wats 11 l Iolos Data
Illumination - soo-watt lamp Plate - Wrattcn M Filter C (omllgc) 1':xposlIr(' 4.S S(,t'oluIN

ANTHRAXBACTERIA IN Pus SMEAR, SHOWINGTHE CAPSULE MAGNIFICATION I700X

The bacillus of anthrax is a fairly large organism, usually conne~ted in short chni."s. Ordinary staining methods do not reveal the presence of the gelatlI10us capsule which surrounds the cells and is responsible for the adherence 111 chain fashion. III taking this micrograph, special attention was given to emphasizing the bn '[cri:! and their capsl:\les and subordinating the pus cells. Exposure Data Objective - r.s mm. (r zox) apochromat l~y piece - Zeiss #8 compensating Condenser Watson. I l olos . Slid' stained with Negri body stain Illumination - S ampere arc lnmp Plate - Wrattcn M Filter - E (light red) Exposure - 1 second

330

PLATE

XXIX.

J\ilE:-l"INGOCOCCUS, SHOWING NUCLEUS AND CAPSULE J\ilAGNIFlCATION 4500X

PLATE

XXVIIT.

TREPONEMA

PALLIDUM, IN SITU I
4100X

HUMAN

KIDNEY

MAGNIFICATIO

The causative organisms of many of our common diseases were identified around the early 1880'S, but science looked in vain for that responsible for syphilis. It was only a scant thirty-five years ago that this corkscrew-like organism was found to be the cause of this dread disease. This seems strange, in the light of the ease with which it can be demonstrated now, as is evident from the micrograph. In this view the organisms are seen in a section of a syphilitic kidney where they occupy all possible positions and are only occasionally in focus. While the micrograph is only an optical section, the blending of the non-sharp portions of the organisms into the plane of focus results in an approximation of three-dimensional effect.
ExpOSU1'e

Bacteriologists h'ave been divided in opinion as to .the pos.sibility of bacterin bcill/-t nucleated. Ordinary staining methods do not. furnish posltlve proof. In the f.II'SI place, the organisms are so small that nuclear differentiation has not seemed posslhh' although there is frequent evidence of a granulated nucleus 111 some of the Iargl'I' species. . . . .. The micrograph shown here is one of the most posltLve proofs, 111 all pJ'ol)l~bJ! 11 y, of a definite nucleus within the cytoplasm of the cell during active prolifcrariou, \~I lease. The meningococcus is extremely small, averaging around onc nucron III dl arnctcr. Stained with carbol-fuchsin It presents a uniform red appearance .Wlll'" examined visually. When photographed at a very high power, uSing the J) 1111('01 the electric sodium lamp as the illumination source, ,not. only a centrally plnn'd nucleus is evident, but a capsule also, the presence of which ISnot susp cc .d l~rhl'I''.1'1 I', It is difficult to bring these out !11 a rcprodu .tron as rhe~ show on the 1~l'lIll' 1 ill' I' .nson for rh' differ ntiation lies ill i hc gl' 'nlCl' nbsorpt inn of the J) mv h t1H' 1I\1~'1 'liS, The nu ,I 'us averages :11>0111 ,l IlIit'I'OIl ill diameter. /';"/1111/1/'1' / )11/,' l'II'(,ll'i(' \lIl1illlll , 111111. 1,1 .A. ,q"lt 11111111.11 11111111111,1111111 lld, \'I' 1'1.11 I' \\'1 ,1111'11 1111111 d 1\' I'\I'PII'II' ,\, IlpI.III.II. uih d I' I'" lilt' J I 1111111111 ( 111111<111'1 I I
()III"\'II\
l'

Data
Illumination10 ampere arc .larnp Plate - Wrattcn Panchromatic Filter - E (light red) Exposure 10 seconds

Objective - 1.5 mm. (I20X) apochromat EyepieceZeiss #8 compensating ocular 'Condenser - Warson Holos, oiled Section stained with Fonrnna's method

"I

I.lIl1p 11111)1'

332

PLATE PLATE:

XXXI.

TRYPANOSOMA

GRA

ULOSUM

JVIAGNIFlCATION 2500X

XXX.

SPIROCHAE:TA OBERMEYERII
2500X

MAGNIFICATION

Here we have another illustration of the value of high magnification in depicting minute organisms. At the magnification employed for this micrograph, details within the bodies of the spirochaetes are clearly evident. While they would still register on the plate at a magnification of rooo diameters, they would be relatively inconspicuous and probably not discernible to the unaided eye. This is the organism which causes relapsing fever. Its minute size is appreciated by comparison with the size of the red blood corpuscles. In photographing a subject such as this, it is important that the organisms be in perfect focus, which generally means that larger objects, such as blood cells, are more or less Out of focus. Exposure Data Objective - 2 mm. 1.4 .A. apochromat Eyepiece - Homal IV Condenser - 1.4 .A. aplanat, oilcd Blood smear stained with icrnsa stain
I

This high-power picture not only provides an easily interpretable view of a trcpan osome, but also illustrates a condition often encountered when using high-apcrturc lenses. At first glance it might seem that a noticeable amount of fuzziness is present, sug gesting a marked degree of empty magnification. But this is not the case, The oh. jecrive was focussed on the fiagellum, examination of which will show it to be sharpl y defined. Nearly everything else in the field is more or less Out of focus, which is Ihe true cause of the lack of sharpness. It is apparent that high-aperture lenses posscss practically 110 depth of focus; even a mall fraction of a micron makes an appreciable difference in the sharpness of lilt' image. In cases of the kind shown here~ one must either attcn:pt the best C()~lIpl'O. mise focus or confine attcnnon to some individual part of maximum mtcrcst, Ignur iog everything else,

Illumination - soo-watt lamp Plate - Wrattcn M ' Filter (ornng ) Expo urc I I1liJ1Ul
1

Esposurc Dnta
Ohjt'('1 ive
111111 l 111111, 1.+

, npochro

Illnminnt
1'1,11('

ion
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I() III11Pt'J'l'

nrc lump
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I.,

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11111

I\'

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Pi"

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1.1111101

PLATE

XXXII.
MAG

PLATE HISTlOCYTES IN BLOOD IFICATION 1300X

XXXIII.

GIA'T

CELL SARCOMA
600x

MAGNIFICATIOr

Although this micrograph has been included here as representative of the general field of hematology, it is so spectacular in what it depicts that it deserves special attention. Histiocytes are rare giant amoeboid cells that in certain blood diseases get into the blood stream. They are so cannibalistic that they not only eat the red blood cells but the large white cells as well. In the picture, the smaller individual at the top is seen in the very act of ingesting a red cell, which is only partly surrounded. The white cells, known as polymorpho-nuclear leucocytes, are often called the. policemen of the blood, for it is their function to destroy all undesirable foreign matter. This they do by eating it up, a process known as phagocytosis. But they cannot get rid of the histiocytes in this manner because th~, latter"eat th~m .1l1stead. The large histiocyte in the ccnter of the field has eaten a pol y and IS dl,gestlI1g it. The clear ring around the poly rep1'esents the degrec to which the digestion has taken place. Large pseudopodia arc to be seen on the histiocyte.

The most prolific production of photomicrographs is to be found in the ~:l(! (~f pathology, but probably in no other field is so much mediocre work donc: I Ills IS not to be blamed 011 the apparatus, but on two controllable factors. One IS rhc U~(' of filters not-suitable to the subject (usually too concrasry) , the other IS failure ~o employ critical illumination, with substantially full aperture. Reduced apcrtur '. III thc illumination cone IS not desirable 111 this class of work where diffcrcnti.u 1(111 is secured by staining.' . . . Pathological an I histological tissues should be portrayed along the lines illustratcd by this micrograph. The nuclei of the cells should not be black, l1n~ess they :11'1' \ isually vcry dark in the section, and there should be no diffraction evident arounr] the various structures.

Exposure lata
Objective K 111111. (20.'-) nJlo('ilrolll:1I I'~)'l'jiiccc I lomnl I (:lIl1d"IIS"I' I." ./\. :lp!;ll1,JI llluuunut illll illll \\,111 1.1I11j' S," 111111 "I,IIIII'd \I iliI 111111.1111 \ 111\.11111111 ,Ill P1:Jl' Wmucn M Filler Il (~I'el'n) FXJlo~III'l' lO "1'('OIHI~

Exposure
-Objective - 2 mm. 1.4 .A. apochromat Evcpiccl' l lomnl IV CCl1ldl'IIWI' \\'.11'011 1111111 , "ill'd 111110.1 ,1111',11 1.111IId1\ illl (;11111" ,1,1111

Data
1I111111il1<1liOIl,00 wntr lamp Plait' \\lnlll'lI 1'1 Filll'l' J! (Iif-tlll rrd ) I' '1"1\1111' I "'llIlId

117

PLATE

XXXV.

FERIUTlC CAST IRON

JVlAGNIFICATlO. 300X PLATE

XXXIV.
MAG

GRAPHITE I.

CAST IRON

IFICATlON IOOX

The cast-iron metallurgist is much concerned with the nature and dispersion of graphite in the metal. Cast iron usually contains around 30/0 to 3J% of carbon, only a small proportion of which is combined with the iron, as in steel. The balance is present as graphite. Large graphite flakes tend to lower the strength of the metal; hence the desire to obtain small flakes and a uniform dispersion. ~hen exarr:ining a specimen of cast iron for graphite, the surface must be highly polished to eliminate scratches, but It IS not etched. The graphite then shows as black flakes against a mirror-like background, the metal itself, Examining and photographing metals requires the use of vertical illumination and objectives which are corrected for use without a cover glass, Exposure Objective - 16 mm, (roz) apochromat Eyepiece - Hornal II Vertical illuminator with plain glass, 10 ampere arc lamp Specimen unctchcc' Data Plate - Wrattcn M Filter - Wrattcn C (blue-violet) Exposure - 1 second

For examination of the matrix of cast iron it is necessary to subject the polished specimen to an acid etch which will attack the constituents differently and thus reveal the structure, This micrograph shows the appearance of a: low-grade cast iron in which practically all the carbon has become graph.ite and none is left in combination with the iron, This not only results in excess gnphite (the black areas) but lowers the strength of the metal, .which is materially stronger when it has the p1'Opcr pl'r centage of carbon combined with it, as in steel. All metals when examined.under the microscope are seen to be composed of indi vidual crystal grains, These grains are clearly evident in the micrograph, In Ihis case they are called [errite grains (i.c, meaning pure iron), whereas if the propl'r a.nount of carbon were present in them, they would be known as pearlite grains, Exposure Obj ctivc 16111111. (10.1:)apochrolllat I')' 'pi, 'C l Iomnl I \ vrt icnl illuuiimuur wit h plnin f{1.1'~. 10 :lllll'l'IT nn' 1:1111p SI'I'cillll'll 1'11'1ll'din I'~ nil." Data Plarc Wrntrcn M Fiht'r \Vr:lll '11 #64 plus l) (pllrplt-) F\IHI~lIrl' 11 S,t'Oll(ls

lll)

PLATE XXXVI.

SPHEROlDIZED CAST
r

Ino
PLATE XXXVII. PEARLlTE GRAIN IN CAST IRON

MAGNIFICATIO

2000X

Carbon :which is. combined with iron .in steel and cast iron forms a compound known as iron carbide, or cementite. This occurs scattered through the grains, usuall y as thin parallel plates, as though the metal were laminated. Sometimes, however, it exists in s~all globules, as seen in this micrograph. As these globules are often qUite small, It reqUIres a lugh magnificarion to reveal them and the nature of their dispersion. High-power metallurgical micrographs should show minute constituents, such as these, sharply outlined; the entire area of the picture should be evenly illuminated and m focus everywhere. To meet this latter condition, the surface of the specimen should be at absolute right angles to the optic axis. . Exposure Objective - 2 mm. 1.4 .A. apochromat Eyepiece - Homal IV Plate - Wratten M Specimen etched in 3 % nital Data Vertical illuminator with plain glass, 10 ampere arc Ia 111 p Filter - C (bl ne-violet) Exposure - 35 seconds

MAGNIFICATION 3000X

Extremely high magnifications often can be used to advantage in me~allographi.c work. Structures which otherwise might escape detection are made evident. It I~ especially desirable that all unusual metal structures, new alloys, ctc., be sl.11l jected to high-power analysis, for in this way new facts may be gleaned rcganhllg them. TI-iis micrograph shows all example of high magnification applied to the pen rl il i(' structure of a coarse cast iron. It includes a portion of four grains. The laminu oC the cementite plates run in different directions in each. Exposure Objective IIlnt 2

Data Vertical illuminator with plain glass, 10 aJ1Jpere arc lamp Fihcr C (hlu i-viole ) Jo:.~poS\lrc I minute

mm.

1.4

N. . apochro-

]':ycpi 'cc l lomnl IV Pial' \ Vrnu cn 1'1'1 Specimen etched ill auuuonium

Pl'l"\ltlphlllc

PLATEXXXVIII.

E~ERIMENTAL WELDS ON STAINLESS CLAD STEEL MAGNIFICATION 8x

PLATEXXXIX, SERPENTINE ROCK SECTIO !lAG!IFICATlON 20X Rock sections offer problems' of an unusual nature to the photomicrographer in rhnt t~y require ,polarized light to reveal the different minerals present, Visually Ill, result of examination under polarized light is very satisfying, at least from an aesthetic viewpoint, but to translate all the beautiful effects seen into a black and white pie ture is quite another matter. Color intensities as they 'affect the eye are often at variance with the extent to whichthcy record on the sensitized plate, Another condition prcscnt is the eXIinction of crystals with crossed prisms which causes them to appcar black in the cxrincrion position, whereas they are actually n'ansparent and possibly highly colored ill other positions, f\ serpentine rock, such as shown here, is Icss difficult to portray than 111:111)' Oill('I'S, he .ausc thc mesh structure of thc altered olivcnc crystals and the alteration producis of other minerals posscss some pattern which ';111 be reprodu .cd in black and white, "1','11under .rosscd prisl11s. ",\/)(111/1(' '),,1(/

Unusual problems frequently confront the metallurgist in connec~ion with ?is constant search for improved metals, This micrograph shows the pIcture which was made as a permanent record of the relative quality of three different w:lds on a stainless clad steel. In this case the question was not one of strength - this must be determined by another kind of test - but of the relative resistance to acid attack on three different weld materials, and the effect of the welds on the structure of the ad jacent steel. It was therefore necessary that all samples be treated exactly alike, They were all embedded together, for this treatment, within a suitable matrix and handled as though thef were one piece" '.The notches ?n the e:lds of the bars were for the purpose of identifying the individual samples m the micrograph. , This is an illustration of low-power metallographIc work. In this case a special form of O'lassreflector is mounted under the lens, i.e. between the lens and thc SPCCImen, as ~ertical illuminators are not available for mounting above the largc Planars, Exposure Data Plate - Hammer, low Fill 'I' 4-il1('h K 1 J!XPOMII'(' I ~('('(IIld

Objective - 75 mm, Planar , Illumination - 10 ampere arc WIth spccial plain glass mirror

Ohit'('1 ill' SO 111111. 1'1.111,11' ('''II(kll~''1 ('Ill, \111 iI 1 1111 iI 11 "I 1'1.111' \\ 1.1111'11 \1 1.lIltl I Ill' iI .dlll1 1',,1.1111111 I'"

Hhuuiu.u iun I 1'" 111"

SOO \\1111Illlllp "'('(111(1

lH

PLATE XL.

DIORITE DRIFT ROCK


22X

MAGNIFICATIO

PLATE XLI.

C01'TON HANDKERCHIEF, SHOW MAG IFICATION


20X

BY OBLIQUE Top

LIGHTI G

This micrograph illustrates what can usually be expected in the portrayal of rock sections where practically every field ~'Ill possess one or more crystals 1I1 a position of extinction, no matter what the rotational angle ma~ be. . Careful adjustment of the rotational posltlon JJ1 this case has all~:ved ~ .conslderable portion of the plaglOclase feldspar and the hornblende to appear m parnallllumlnation, at least. Occasionally, when an excess of crystals occurs 111. an extension POSItion, no matter what the rotational position may be, It IS pel"llllsslble. to uncross the prisms slightly. This should never be done, however, when It IS desirable to reveal the presence of isometric. crystals. .On the other hand, O~1eshould trr to e.ll.mll1ate the presence of all anisctroprc cryst~ls m an extinction posltlon whe~ It IS desl1e,~ t.o concentrate attention on those which are to be Identified by then non-polarization.

. Pictures of textile fabrics at low magnification, taken with oblique top illuminu non, do not present any unusual problems to the photomicroglapher., Most impor t~nt IS .the l!glltlng; this might reqUIre some slight modification 111the angle of t lulight WIth respect to the optic axis, or the azimuth direction with respect to the WC:1Vl', for each type of fabric. The most. important requirements arc to accentuate I Ill' weave Itself and to bnng out the individual fibers of which the thread is COl11 pos('d, . To the uninitiated, a pIcture such as this provides a better appreciation of the 111(':111 JllO" of magnification than almost any other subject. There is always surprise 11I:l1li fcstcd when It IS pOlllted out that the magnification is only twenty times. '1'11(' average guess puts It anywhere from onc to several hundred times.
J<:X{iO.l'III"

Exposure
Objective - 35 mm. Planar . Condenser - Spectacle lens with larizer Large abbe prism in tube, prisms po-

Data
Plate - Wrattcn Pnnchroruatic Illumination 50o-wall 1:1I1lp KxposlIre I, sl"t"olld

Dnt

Objective 5111111. PI:lIHII' UI F:!) Hluminut ion i 11111111'1'1' .ur, \I II\J"III

crossed

Pint( "'1":111('11 Filll'l' 11I1IH' ('lIl1illHlIilll' I'IlIldl'l1~I'1 1 11'''111"1' "'\'r'lIld'. F,II1I'1I' 'IIPI1"III'd ,11111111 1:-.11111\tllllllltl,\1I,IIIIII'lllllllId

344

PLATE XLIII. PLATE XLII. SQUILLA DESMARESTIl, SHOWN IlY DARK FIELD TLLUMINATlO

NOSTOC, SHOW

BY DARK FIELD ILLUMI

ATlO

MAGNIFICATION 475X

MAG, lFlCATTON I2X

This beautiful animal, the alima stage of Squilla desmarestii, offers a fine subject for low-power dark field. In his case the dark field must be secured through the use of a central stop in a Iow-apertured condenser. Standard dark field condensers are designed for high-power work and cannot cover the field required for such a large object. The considerable depth of the body of the specimen required the use of a longfocus lens to accommodate it and show every part in focus. Exposure Data

Objective - 50 mm. Planar slightly Condenser - 14 .A. aplanat, with stopped top removed and central stop below Filter - E (deep orange) Illumination - 50o-watt lamp Plate - Wratten M Exposure - 5 seconds Object mounted in fluid, without pressure, not stained

Thi~ minute blue-gre~n alga, only a little higher in the scale of life than the bnc terra, .IS.a beautiful sU?Ject for d~rk field work. The minute coccus-like spherical cells, J?lI1ed together Il1 chams, simulate fairy necklaces as they glisten in the hril liant light of dark field illumination, The specimens shown here were mounted ill fluid and hence are not all on the same plane. Even with the use of a lowcr-ap .r tured o~JectIve not all could be 111 focus at the same time, but the results are suggestiv , ~f a three-dimensional plctu.re, for the eye Itself can focus upon but one. plane ut u time. The micrograph is typical, .otherwise, of dark field work. The only problem of an unusual nature lies JJ1 obtaining the suggestion of translucency in the cells rnrher than a brilliant whl~e, devoid of detail. This is clone by working [01' softness, rather than contrast 111the negative.
/IX/lO.\"IIY(J

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PLATE XLIV.

TOU-LE-MOI

STARCH, SHOWN BY POLMIZEO LIGHT PLATE XLV.

MAGNIFICATION r60x

Rrcxsrs

RAT JOINT, SHOW

BY INFRA-RED LIGIlT

Starches are easily identified in microscopical preparations. by means of polari.zed light. With the polarizing prisms crossed, the background IS dark, but any object possessing birefringence, which. may be in the field,. appears !llumll1ated due to the interference set up by repolarization, Starches, being 111this class, show bnghtly against the black background, each gl'ain marke~ with a black cross. The arms of the cross connect at the hilum, or core, of the gram, which In most starches IS not centrally located. Tou-le-moi starch grains are among the largest known, and ther~fore serve well to demonstrate the typical appearance of starch under polarized lIght. In takmg a micrograph such as this, the exposure and development should be correct, so that the grains will stand out brilliantly white, on a black background. Exposure Objective - 16 mm. (rox) apochromat Eyepiece - Homal T Condenser - 1.4 . A. nplan;1t Polnrizcrs crossed Data Illumination - soo-wall Inll1p Plate "Vratt '11 1\ I 1':"poslIrc .H Sl"t'OIHis

MAGNIFICATION rSx

The value of infra-red light in the photography of some types of tissue is wcl I brought out in this micrograph. The section, cut in celloidin, was nlOu~lled III~ stained, in glycerin. This necessitated the use of the microscope in the vertical PO,I tion.' Comparative tests with light in both the visible and ultra-violet regions d'1I1011 strated conclusively the superiority of the infra-red in this particular case" Infra n'd brought out structural details not made evident by either of the other light hands, Information also is gained which cannot be secured by standard staining III '(hods, Exposure Objectiv 20 Pia "'1 I' , Illuminat.ion .l()O-W:1I1 1:IlIlP, Wilh I iihkl' 'I'Sll'lIl COlldl'IISl'I" Z 1'111. '1l\'('LIl'\to II'!\'. [Iat a
Fih.cr Pluic Schou infrn-r 'd

I':astlllan Sp 't'll'Ogrnphic #1 I' ItxpllslIl'l' H Sl'l'lIl1ds

PLATE XLVII. PLATE XL VI. V ANA))IUM DISPERSION IN VANADIUM MITrE)) ULTRA-VIOLET CATALYST, SHOWN BY TRANS-

FROSTED SURFACE OF ELECTRIC LIGHT BULB, ILLUMl:-<ATED BY INCIDENT LIGHT , J\iIAGNIFICATlON IOOOX

This frosting

LIGHT 275 W,L.

MAGNIFICATION

80x

One. of the most valuable contributions of the ultra-violet microscope to science has been 111 the Ide~tlficatl?n of various constituents in a given specimen, through '1 difference 111 their relative transmission of ultra-violet light. This micrograph is :I typical example. The problem was to determine the dispersion of the vanadium compound 111 a catalyst pellet. Was it uniformly distributed throughout, segregated, or otherwise? By ordinary methods nothing could. be ascertained; special methods to differentiate the vanadium were necessary. A section (about 15 microns thick) was photographed by an abnormally long exposure, to over-expose all constituents 110t vanadium. The latter is so absolu~ely op~que to ultra-violet that even an exposure of several hours :vrll not make an impreSSIOn on the plate, where even a minute quantity of vanadium IS pre~ent. Hence, we have obtained a silhouette picture, the blnck I' 'prCSl'llI ill!\, vanadium, the white, all other constituents. A normal exposure would have shown structure in the non-vanadium constituents. l\.1'/J
0.1'11

object offers photomicrographic problems of a very unusual nature, The is done on the inside of the bulb, every part of which is concave or COI1l positely curved. The etch is very fine, requiring high magnification to reveal ils nature, and two types of etch are present - pyramidal elevations with flat angular sides and a secondary structure in the crevasses, consisting of rounded depressions, Thus the total depth of focus required is considerable, Lastly, the material is transparent glass. The illumination must be fair! y symmetrical, to avoid deep shadows. In one respect the micrograph requires explanation, that the true nature of rhc rounded areas may be understood. At times they give the impression of rounded elevations, whereas they are cavities. The reason is that, being glass, the reflections from a negative SI here can be identical with those of a positive sphere, In a 1110IH)(;ular view there is no way of distinguishing them apart. U~'I}(J,wrc Dnt Zl'iss Epi \ V Cllnd('llM'I',

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011

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PLATE

XLVIII.

PAINT TEST PANEL


FULL

0 W

e-

0:
f-<

MAGNIFICATIO

SIZE

if)

Research departments of large manufacturing concerns are continually making tests of products under exaggerated service conditions, with a view to establishing causes of failure and the average length of life to be expected. To supplement the written records of such tests, it often adds materially to the data to attach a picture or photomicrograph which visualizes the appearance of the product after test. This picture shows one of a series of paint panels after going through a specific routing of conditions. From the standpoint of magnification, the picture represents the borderline between commercial photography and photomicrography, as the subject has been neither enlarged nor reduced, in the taking of the picture. The paint on the panel was white, but the color not being a factor in this case, it was allowed to appear appreciably dark, in order to emphasize the presence of the small blisters, the cause of final failure. To be of real value, a picture of a surface such as this, must suggest a three-dimensional aspect. This is accomplished entirely by manipulation of the lighting. Exposure Data Filter - none Objective ~ 7-inch Dagor , xposure - I second Illumination - one 4oo-watt lamp Plate - Hammer Slow Light very oblique to surface and far away, to giv .vcn illumination, was well lighted by laylight, in addition to nrt if 'inl light.

~ ....J ~

-< c,

e -e

The

J'()OIlI

352
INDEX
Abbe condensers, 19, 20 theory of resolution, 36 Aberrations in lenses, 6, 17 Absorbed light, 135 Absorption of infra-red rays, 76 Achromatic objectives, 14, 17, 19 use of green filter with, 242 Acid hypo, 257 Action of light on silver salts, 254 Adjustment of mirror with vertical cameras, J 14 .Agfa color process, 227 and Lumiere screens, compared,
228

Arc lamp floor stand, 74, 18 ( Arc lamps, 69, 70, 7 1,93, 204, 2 ( ( Artifacts in object, elimination of,
164

Astigmatism, 10, 208 Attached cameras, 42 Author's method of critical illumination, 192 Autochrome color process (Lumiere), 226 Autofocus enlarger (Eastrnan), 260, 269 Basic exposure time, determination of, 154 Bausch & Lamb amphiplancyc pieces, 80 condensers for low powers, 62,
65

Airy disc, 8 Albumen emulsion, 247, 2)2 Alignment of optics, securing, I 12 Alpha lantern slide plates (Ilford),
247 Amidol, 273

Arnphiplan eyepieces (B&L), 80 Amphipleura pellucida, test for U- V resolution, 218 Angular aperture, 8, 9 Antipoint, 8 . Aperture diaphragm, 38, 39, 118 variations in low power lenses,
152,.153

mercury vapor arc lamp, 72, 73 metalloscope, ILS, 200, 20 I, 203 micro-tessars, 62, 65 neutral tint filters, 76 ortho-stereo camera, 234 outfits for photomicrography,
50-53, 56, 57

Aplanatic condenser, 20 Apochrom'tic objectives,


80, 205

17,

19,

Apparatus, setting up, I TO for specialized photomicrography, 59 required for photographic proccsscs, 26 ( Appli ations of mi .rophotography, 2.p

simple metallurgical microscop , 198 special photomicrographic camera, 78, 79 tungstenarc lamp, 71,72 ultra-violet outfit, 217-219 vertical illuminator, 197 Bellows, design and construction of,
97, 100,
101

length, effect on focus, ( (9 cffc ton magn if cation, 4, 5,


22

2 (,

354

INDEX

INDEX

Bentley, Warren P., snow crystal pictures, 89 Binocular body, 16 vision, 235 Bispherical condenser, 206 Brain sections, 178 Brownian movement, 206 Brushes, 125 Bull's eye condenser, 3 I Cadmium spark, 275 mu for U-V work, 220, 22 T Camera attachment for Greenough binocular, 234 extension, 127 lens, 2 Cameras, see under specific types Cardioid condenser, 206 Carrier for negative in microphotography, 248 Cause of flare or fog spot, 190, 191 Cell, see Cooling cells Centering condenser, 113 of objective to condenser, 117 Change of temperature, effect on focus, 176 Characteristics of filters, 134, 137143

Collodio-bromide emulsion, 247 Colloidal solutions and solids, photographing, 207 Col or filters, 18 adequate illumination required for, 68 comparison of various types,
137

use of improper, 186 photographs for polarizing objects, 210 photography with Rheinberg discs, 207 photomicrography, 45, 226 processes, 227-229, 232, 233 screens of Agfa and Lumiere plates, 228 Dufay and Finlay plates, 230 work with separation negatives,
232

Condenser, 16, 19, II 3 centering of objective to, 117 for dark field, 206 Condensers for low power photomicrography, 20, 62, 65, 67 mounting of homemade, 93 Condensing lenses, 101 special, 77 Conditions in the object, overcoming, 169 Conjugate focus, 2 Connecting microscope to camera,
99,
!O2

Design of camera bellows,


!OI

97,

100,

Determining the basic cxposur' time, 154 the degree of development, 277 the focus with ultra-violet illumination, 212 the magnification with rninicams,
134

Contax-miflex
45, 110

camera

(Zeiss),

43,

Colorless objects, photographing


170

of,

of light intensity with resistances, 100, !O3 Cooling cells, 75, 76, I I I hornemade, 94 Cover glasses transmitting ultraviolet, 222 Critical illumination, 29, 30, 32, 33,
37,39,118,192

Control

Developer, defined, 255 Developing, defined, 254 and printing technique, 274 and printing, glassware for, 271 solutions, chemicals for, 272-274 formulas, 289-293 tank for film packs, 264 roll film, 263 Development, chemistry of,' 254 determining the degree of, 277 over and under, 185, 276, 2Hz,
283

Checking vibration, 159 Chemical fog, 256 Chemicals for developing


272-274

solutions,

Combined straight and right angle optical bench, 1<)9 Commercial photomicrographic equipment, 41 Common faults in photo micrographs, 184 problems in photomicrography, 165 Comparison eyepiece, 17 of critical illumination methods,
34, 192

time, graph of, 277 Diagram of setup for microphoto

r.,

by imaging the light source, 192 Critical lighting with vertical illuminators, 204 Curvature of field, overcoming, 165 Cut film, tank for, 262, 263 hangers, 26 I, 263 Cutting tool for making masks, 27 I

raphyv z yo
masks in front of plate, 160 stops for dark field, 205 Diaphragm, see also Field dinphragm, Aperture diaphragm Differences in appearance, vertical and oblique top light, 202 Diffraction, cause of circle of confusion, 22 caused by narrow cone of illumination, 189 Diffusing the light source, 95 Dispersion, 7 .Distance of best normal vision, 15 Dufay and Finlay screens, '01;1pared, 230 .olor proccss, 228 Dusring of pin s, 122 Dvcs, t 1';1 I1SI 11 ission .hnrac 'rist ics . Of,'IH,145

Chemistry of development, 254 of fixation, 256 Choosing the proper filter, 160-162 Chromatic aberration, 6, 7, 17 Chromotone color process, 233 Circle of confusion, 8, 22 Cleaning solution for trays, 298 Clocks, see Timing clock, Interval timer Closing diaphragms in low power lenses, 178 Coherent light, 36

D line of spectrum, D-Iog E curve, 274,

2I I

276

Compensating eyepieces, 18 Composition, pictorial, in photomicrography, 163 of developing solutions, 255 Compound microscope, 12, 13 parts named, 14 Computation of exposure time,
157

Dagor len~ 10, I I Dark field photomicrography, 205 ob] ects, photographing, of, 170 Darkroom, 109, 257, 258, 260 Data book for basic exposures, 156 Defender Photo Supply Co. col or process, 233 Density of object, effect of, on exPOSll

Computing exposures with slide rule, 124 Concentrated f lament lamps, 93

r ri me,

152 J

f)

.prh of [ocus, I I,

(lH, I Ho
ZC)!)

Dcscnsitizing

solution,

11

356

I JDEX

INDEX

357

Eastman Kodak Co., enlarger, 266 interval timer, 265, 266 Kodachrome color process, 45, 231 safelight, 260, 262 scales, 264 spectrographic plates, 2 I 5 syphon washer, 268 timing clock, 123, 124 Type V spectrographic plates, 247,2P Effect of camera extension on focus,
II9

magnification, 4, 5, 2 I, 22 density of object on exposure time, 152 eyepiece on magnification, 129 filter factors on exposure time,

'48
foggy 'lenses on exposure time, 153 lens focal length on image size, 3,4 light intensity on exposure time, 147 magnification on exposure time, 151 non-axial illumination, 188 numerical aperture on exposure time, IS! plate speed on exposure time, 148 Effective numerical aperture, 30 Electric sodium lamp (Zeiss), 73 Electronic microscope (RCA) , 237, 238 Elimination of artifacts in objects, 164 extinction in polarizing objects, 209 . ultra-violet light, 146 vibration in apparatus, 108 Elon, 272

magnification, 10, 20, 27, 173, 174 Engraving on glass, photomicrograph of, 21, 22 Enlarger (Eastman), 266, 269 printer, Graflex, 266, 268 Enlarging, 281 Envelopes for storing negatives, 280 Equipment for fluorescence rnicroscopy, 216 low power photomicrography, 6r modern photomicrographic, 41, 123,257 Excessive diffraction effects in photomicrogra phs, 189 Exposures, darkening of room for making, 158 over and under, 185,282 use of light meters for determining, 158 Exposure time, 90, 154 computation of, 124, 157 effect of density of object on,
Ip

Empty

micrometer, 123 scales, superimposing 176 Eyepoint, IS Factors

on picture,

filter factors on, 148 foggy lenses on, 153 light intensity on, 147 magnification on, 151 numerical aperture on, IS r plate speed on, 148 factors influencing, 146 Extinction in polarized objects, elimination, 209 Extra long bellows, 97, 100, )01 Extremely high magnifications, 173 Eyepiece (see also Ocular), 12, 18, 125, 129 comparison, 17 compensating, 16 Homal, 80, 132 Huygenian and Ramsden, 17 large diameter, 13

governing magnification, 127 influencing the exposure time, 146 Farmer's reducer, 295 Faults in photomicrographs, 184 Ferrotype plates, 270 plate wringers, 270, 271 solution, 298 Ferrotyping prints, 281 Field, defined, 127 diameter, 127 diaphragm, 36, 38, r 16,120 Film, see Roll film, Cut film, Film pack pack, developing tank for, 264 Filter factors, Wratten M set, 137, 138 effect on exposure time, 148 holder, design of, 93, 94 solutions, 76, 136 Filters, characteristics of 134, 137143, 148 comparison of various types, 137 for elimination of ultra-violet, 146 for infra-red, 214 for use with mercury arc, 212 J ena glass" 137 kinds of, 136 neutral tint (B&L), 76 use of improper, 186 Fine grain developers, 273 plates for microphotographs, 246 Finlay and Dufay color scr ens,
2jO

Fixation, chemistry of, 256 defined, 257 Fixed mirrors, I 15 Fixing solutions, 293, 294 Flare or fogged spot in micrographs, 190, 191 Flat field oculars, 80 . Floor stand for arc lamp, 74, 18 r Fluorescence defined, 214, 215 microscopy, 55, 60, 2 '4-2 16 Fluorescent glass plates for focussing U- V image, 2 I 2 Fluorite objectives, 17 Focus, conjugate, 2 depth of, 18, 168 , determining, with U- V illumination, 2 r 2, 2 13 principal, 2 Focussing glasses (magnifiers), 12 . mount on lens board (Zeiss), 7n with reading glasses, 16o Fog, chemical, 256 Foggy lenses, effect on exposuJ" time, 153 Formulas, 288-299 F otoflood lamps, 71, 72 Freedom from dust and fumes desirable, 19 vibration desirable, 90, 176 Funnel tube for mounting large lenses, 66, 69 Gamma, development factor, 276 infinity, 276 Gelatin filters, 136 Glass filters, Schott & Gcn, jcna, 136, 137 . lenses, degree of cone .tion ill ul tra-violet region, 212 transmission characteristics, 212 ;Iass\ :11" for photo l'l'nphic prot'-

.olor 'pro

'CSS, 22<)

sscs? l70

INDEX

INDEX

C)

Glycin, 272 Graflex enlarger printer, 266, 268 Graph of development time, 277 D-Iog E curve, 275, 276 . Graphing of magnifications of optIcal system; 133, 134 Green filter, with achromatic objectives, 204 line of mercury arc, 2 I 2 Greenough binocular, camera attachment for, 234 Ground and opal glass, transmission of, 194, 195 for diffusing light source, 95 Hammer slow plates, 12 I Hand mirror for focussing by distant control, 123 Hanger for cut films, 261, 263 Hardening baths, 294 Homal oculars, 80, 132, 166 Homemade cooling cell, 94 equipment, 8 I requirements for, 85 Horizontal cameras, 87 -vertical cameras, 52, 54, 56-59,

96
Hydroquinone, 273 Hypo (hyposulphite of soda), 257

Ilford alpha lantern slide plates, 247 Illumination, 28, 89, 187, see also Light, Lamps, Vertical illumination and Incident light critical, see Critical illumination, K6hler illumination equipment for, 68-75 requirements for ideal performance, 40 system for metallographic work, lO4, lO5 Illuminators, vertical, critical lighting with, 204

Illustrative photomicrographs, (plates) 300-352 Image, virtual, 2 size, relation to object size, 2 Imaging of light source, 119, 192 vs. light condenser, 192 Immersion oil bottle, Zeiss model, 124, 125 Improper color filters, use of, I ~6 Improvised shutter for controlling exposures, IQ3 Incident light, 179 Incoherent light, 36, 37 Indirect illumi nation, 179 Infinity, object distance, 2 objectives corrected for, 19 Infra-red, filters for, 212, 214 . rays, absorption of, 76 Inherent limiting conditions in photomicrography, 171 Intensely stained objects, photo. graphing of, 170 Intensification of negatives, 282 Intensifying solutions, 297 Intensity of illumination, resistance control of, 100, lO3 Interference figures, photographing of, 210 maxima, theory of, 26 Interval timer (Eastrnan), 265, 266 Inverted metallographic micro.scope, 199 Jella glass filters (Schott & Gen), 76, 137 transmission of, 141-143 Kits for small size plates, 122 Kodachrome color process (Eastman), 231 miniature cameras, adaptable for, 45 Kbhler illumination, 33, 34, 36, 37,

39, 75, 116, lI8, 119, 177, 178, 192 see also Critical illumination Labyrinth, darkroom entrance, 258, 260 Lamp housing and negative holder for microphotography, 248 Lamps, operation of, I I I (safelights), for developing, 263 various types of, 69-74, 89, 93,
2I I

Lantern slides, 284-287 Large research outfits for photomicrography, 41, 53 transparent sections, photographing, 178 Latent image, 254 Le Chatelier metallurgical microscope, 57 Leica camera, 42, r lO manual, 110 Leitz, leica camera, 42, r lO macro camera, 47, 48 -ibso attachment, 42, 43 -ring illuminator, 75, 76 milars, 65 panphot, 55, 57, 6r photomicrographic cameras, 5 r , 55 sliding focussing attachment for leica, 44" sum mars and micro-sum mars, 65 Lens board mounting for lenses, 63, 66,68 camera, 2 positive, 2 Lenses, see also Condensers, Objectives, Eyepieces ab rrations in, 6 for low pow r pi1otol1lnnog rnphv, 1_ suituhk- for mi T()plt()tll~I\;lplt\, 1./-1

see also Polarized li dlt, Lamps, Ultra-violet, lilt 1':1 red, Monochromatic action of, on silver salts, 2S-I intensity, resis~ance control of, lOO, lO3 Light meters for determining .~ posure, 158 source, diffusing with ground glass, 95 effect of, onexposure time, 47 sources for infra-red, 214 transmitted, 135 trap, 99, 102, 110 "white, 135 Limelight, for illumination, 69 Limit of resolution, or useful Ill:lg nification, r o, 20, 22, 2 il, l7 Limitations of minicams forphot () micrography, 46 Limiting conditions in phoromi crographv, 171 Liquid color filters, 136 transmittance of, 140 Living organisms, outfits for II~(' with, 44 Loading of plate holders, IT r Low power photomicrograph wit h incident light, I79 transmitted light, I77 Lucite, for illumination purposes, 105 Lurniere Autochrorne color prol' ess, 226 and Agfa color screens, 22H Luminescence microscopv, 21.j" 215
Light,
C

Macro-a ttach 111 en t, (Lcirz), -I), ~H -ring illuminator (I,citi'.), 7S, 76 j\la rncsium spark for JlloJ1oclmlmnt i uh rn viokt, ZZo,." C 1\1:1 Il1i(ic:1ti()l1, ICI, Z 1, IZ() czH, Il',
171,
I , 17()

1-

INDEX INDEX

determining, 132 with minicams, 134 effect of eyepiece on, 129 exposure time, 151 empty, 10,20, 27, 174 extremely high, 173 graphing of, 133, 134 range of objectives, 126, 128, 131 table of, 131 Magnified image, 3 Magnifiers for accurate focussing, 159 Ma P pi ctures, sectional, 172 Mask templates, 271 Masking prints for white borders, 279, 280 Masks, diaphragm, in front of plate, 160 Materials required for photogra phic processes, 254 Maximum useful magnification, 175 McEwen, Alfred, writings on glass, 21 Mechanical stage, 15, 80 Mercury vapor arc lamps, 72, 73,
2I 1

filters for use with, 212 MetalIographic plates (Wratten), 205 Metallography, 60, 74, 87, 196 special illumination system for, 104, 105 Metalloscope ILS (B&L), 200, 201, 203 Metallurgical microscope, inverted type, 199 Method of making microphotographs, 248 Meters, see Light meters Metal, 272 Micrograph, I MicrOI~eter, stage, 123 eyepIece, 123

Micron, defined, 7 Microphotograph, I Microphotography, 241 lenses suitable for, 244 plates for, 246 practical applications of, 242 Microscope, compound, parts named, 14 Micro-surnmars (Leitz), 65 Micro-teleplats (Spencer Lens Co.), 65, 66 Micro-tessars (B&L), 62, 65 MiAex camera (Zeiss), 48 Mikrotars (Zeiss), 66, 67, 68 Milars (Leitz), 65 Millimeter scale, 124 Miniature cameras in photomicrography, 42 Mirror, adjustment of, for vertical cameras, 114 hand, for distant focussing, 123 Miscellaneous equ ipment, 123 Modern photomicrographic equipment, 41 Monochromatic light, 7, 2 ro, 212, 217 quartz optics, 220 Monomethylpararninophenol, 272 Motion picture photomicrography, 44,60,223-226 M-Q developers, 273, 274 M-set of Wratten filters, 136 Multiplication back for test exposures, 77, 78, 154, 213 Narrow spectral regions, photographing with, 210 Near ultra-violet, photographing with, 212 Negative envelopes for storage of plates and films, 280 eyepieces, 18 egatives, spotting and rctou 'hing, 271

erst lamp for illumination, 69 eutral tint filters (B&L), 76 transmission of, 194, 195 on-axial illumination, effect of, 188 umerical aperture, 9 effect of, on exposure time, 151 relation to resolution, 25, 26 Objective, 12, 16, 80, 125 see also Achromatic, Apochromatic, Semi-apochromatic, Fluorite Objective and condenser, centering to. each other, 117 eyepiece combinations, covering capacity of, 130 magnification of, 14 Objectives, corrected for definite tube length, 18 designed to work without cover

glass,

202

transmission charactcrisi i('~ 11(, 194, 195 Opaque objects, illumination fOI, 74, 75 . Operation of lamps, I I [ Optical alignment, securing, I I bench (Zeiss), 76, 95 straig:ht and right angle, '99 correction of glass objectives III u-V region, 2 I 2 plane, 1 I, 12 sectioning, 167 Optics, quartz, for monochromat i ' U-V light, 220 Ortho-stereo camera (B&L), 234 Overcoming conditions in the oh ject, 169 curvature of the field, 165 Over-development, 195, 276,. 2H Over-exposure, 185, 282 Overlapping of magnifications 01 objectives, 128 Paget color process (Finlay), 22<) Palm rotary print washer, 270 . Panchromatic plates, 120, 12I, 276 Paraboloid condenser, dark fjeld, 206 Path of rays in cardioid condenser, 206 Pedesis, 206 Phosphorescence, defined, 21-+, 215 Photoflood lamps for photomi 'l'og'raphY,93 Photograph, [ Photographic equipment and nppil rarus, 2S"j., 257, 201 Phoromn Togr~l ph, I Pil()lOlIli('l'ograph, I PilOllJlllit'l'o 'r:lphi' 'l["iP"It'1l1, 41 (or ~p('('i:li p" I'pO~t'\, ~I) IIICI(IlIIII'l'llt'I':lp".\ III 'Idor, . ~()

overlapping of magnifications, of, 128 series of, for complete range of magnification, 126 used without eyepieces, 18, 19 Objects, colorless, photographing, 170 dark, photographing of, 170 elimination of artifacts in, J 64 mounted in fluid, photographing, 177 ' overcoming conditions in, 169 stained objects, I35 . with low refractive index differentiation, 170 Oblique and axial top illumination, differences in, 202 top illumination, '79 Ocular, '2, 10, Ho, 100 Oculnrs, (lal fit-Id, Ho, 1 p, 1 M Opal "ass fo), di(rll~ill , litrll1 ~IHII'lT,
I)

--

--~-

-----

INDEX

I JDEX

Preliminary considerations in phoin the infra-red, 214 tomicrography, 108 ultra-violet, 60, 2 I 2, 220 testing of equipment, I IS of objects mounted in fluid, 177 Principal focus, 2 with D line of spectrum 2 I I Principles of critical illumination, incident light, 179 29 polarized light, 208 photomicrography, I Rheinberg discs, 207 Printing by proj ection, 28 r Photon, 272 Printing frame, 266 Pictol, 272 machines, 266~269 Pictorial composition in photopaper (positives), 278 micrography, 163 Print trimmer, 270, 272 Pigeon post films of 1871,241 washers, 270 Planars (Zeiss), 65 Prints, ferrotyping, 28r Plate holders, I I I speed, effect of, on exposure , Quartz mercury vapor arc lamps, time, 148 Plates, dusting of, 122 72, 73 optics for monochromatic U- V for use in the infra-red regIOn, light, 216 214 slides and cover glasses, 222 process, 121 suitable for fluorescence photogRamsden circle, 15 raphy, 217 Range of magnifications of objecmicrophotography, 246 tives, 126, 128, I 3 I monochromatic bands, 2 12, Ratio of image size to object size, 213 2, 3 tanks for developing, 262 ReA electronic microscope, 237 and films for photomicrography, Reading glass for focussing, 123, 120 r60 Point-o-lite lamp, 7 I Record cards for photornicroPolarized light, photomicrography graphs, 124 with, 208 Reducing agents, 255 objects, color processes desirable solutions, 295~297 for, 210 vibration in apparatus, 106 elimination of extinction, 209 Reduction, 255, 282 Polarizin~' films, substitute for Reflected (incident) light, r79 pnsms, 209 Refraction of light, 7 Polaroid, 209 Relation of lens focal length to imspectrophotometric curves of, age SIze, 4 209 negative size to rnicrophoto size, Positive eyepiece, 18 lens, 2, 6 249 Relative coy ring 'flpfl .itv of obprints, 257, 258 [cctiv and cy 'pi ' " '0111Practical applications of rnicrophohinnt ions, 1 10 'tography, 242

.Removal of front lens of substage condenser, I 18 developer stains from the hands, 299 Requirements for homemade apparatus, 85 Research photomicrographic outfits, 53 Resistance control of light intensity, roo, 103 Resolution, effect of anti point size on, 8, 23 formula for, 25, 27 limit of resolution, 10, 20 theoretical aspects of, 20-28 Restraining agent in developers, 256 Retouching stand, 27 r solution, 298 Rheinberg discs, 207 Rib bon filament lamps, 7 I Riders for optical bench, 77 Right angle optical bench, for metallography, 199 Rinsing solution (shorts top bath), 294 Rodinol, 272 Rodol, 272 Roll film developing tank, 263 Rotating stage, 16 Royal Society thread, 13 Safelight (Eastman), 260, 262 Scales for weighing chemicals, 263, 264 " Schott & Gen, Jena glass filters, 76, I37~1 43 , cctional rnap pictures, 172 Sc 'uring depth of fa 'us, 10R, I Bo optical nlignl1lcnt, I 12 sharp foclIs on ''I'OlllHI I'\:l~~,
1 ~()

Separation negative processcs fur color work, 232 Setting up the apparatus, r 10 Setup for microphotography, 250 Shortstop rinsing solution Ior plates, 294 Shutter for controlling exposur s, 103 Sigmar lens, I I Silver salts, action of light on, 254 Simple metallurgical microscope (B&L), 198 vertical cameras, 49 Size of light image in the planc of the object, 35 ' Slide rule for computing expoSlIr 'S, 124 . Slit ultro-microscope, photograph ing with, 60, 207 Slow motion mechanical stage, Ho Snow crystals, photographing, Hc) Sodium lamp, electric (Zeiss), 71,
2I I

illumination system [m: metallography, 104, 105 ph 0 to m ic 1'0g r a p hi c camera (B&L), 78, 79 apparatus, 59 processes, 196 Spectrogram of cadmium and magnesium spark, 220, 221 Spectrographic photomicrograph, 60 plates (Eastman ), 214, 215,2,,1-7, '252 Spectrum,7, I35 Speed of a lens, 9 Spencer Lens 0" low pOWl'1' micro-tclcplats, 65, 66 photomicrographic .amcrns, ..j.C),
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INDEX

INDEX

Spotting negatives, 27 I , Squeegee roller. for ferrotyping prints, 270, 271 Stability of fine focussing device, 176 Stage, 16 mechanical, 80 micrometer, 123 Stains on hands from developer, removll1g, 299 Stereoscopic photomicrography, 60, 234 Stopping down, I I Stops for dark field, 205 Storing concentrated solutions, 271 negatives, 280 Structural detail in dark field rrucrographs, 206 Substage, 16 condenser, 19 removal of front lens of, I 18 see also Condenser Substances not transmitting ultraviolet, 222 Sum mars (Leirz), 65 Superimposing eyepiece scales on micrographs, 176 Symmetrical lens, 10 Syphon washer (Ea tman), 268 Table for photomicrographic outfit, ro6 of magnifications, 131 Taking the picture, 146 Techn~que of developing and print111g,274 photomicrography, 107 Temperature changes, effect on high magnification, 176 Tessar lens, 10,62 Test exposures with rnultiplicator back, 154 ordinary plate holders, 156 Testing of equipment, I IS

Theoretical aspects of resolution, 20 Thermometer for developing tern- . peratures, 265, 266 Thick sections, photographing, 169 Tilting stage for stereo-photomicrography, 237 Time of exposure, 90, 146 Timing clock, 123, 124 Top illumination, r 79 Transmission characteristics of dyes, 144, 145 filters, 137, 140- 143 ground and opal glass, 194, 195 neutral tint filters, 194, 195 ordinary glass lenses, 2 I 2 polaroid, 209 Transmitted light, r6, 19, 135 photomicrography with low powers, I77 Trays, cleaning solution for, 298 for developing, 26r, 263, 266 Trimmer for paper prints, 270, 272 Tube length, mechanical, 13, 19 Tungsten arc lamp (B&L) , 71 Types of photomicrographic equipment, 41 Ultra-Violet outfit of Zeiss, 220, 222 photomicrography, 60, 212, 220 see also Mercury arc, Cadmium spark, Magnesium spark transmitting glass, slides and cov ers, 222 Under-development, 185,283 Under-exposure, r85, 282 Uneven illumination of field, 187 Universal photomicrographer outfits, 55 Unsyrnmetrical lense , ro Unusual problems in photomicrography, 165 Use of extremely high magnificntions, '73

green filter with achrornats, 204 improper color filters, 186 light meters for determining exposures, 158 objectives without eyepieces, 19 ordinary optics in the near ultraviolet region, 2 J 2 quartz optics in ultra-violet photomicrography, 220 reading glass for focussing, 160 Uses of ultra-violet photomicrography, 223 Vertical cameras, 49-55 illumination, 197 and oblique top illumination compared, 202 illuminators, 197 critical lighting with, 204 Vibration of apparatus, 90, ro6, ro8, 176 Vickcrs projection microscope, 58, 63,64 Virtual image, 2 Vision, binocular or stereoscopic, 235 clista nee of best normal, 24, 25 for prints, Eastrnan syphon, 268 Wave lengths of light, 7 effect in resolution, 24, 217 Wavy sections, trouble in photographing, 169 Whirlpool washer for prints, rci8 White border on prints, masks for, 279, 280 Washer

White light, IJ5 Workroom for photomicrography, ro8 Wratten filters, 76, 136, 138, 1 1'_ 143 for infra-red" 2 I 2 #'77 for mercury arc, 212 TvI plates, 120, 276 metallographic plates, 205 panchromatic plates, 120, 12 I, 276

[I

Carl, apochromats, used In the U- V region, 2, r 3 automatic arc lamp, 70, 7 r contax-miflex, 43,45 cooling cells, 76, 77 electric sodium lamp, 73, 74 focussing mount on lens bon I'd, 68,70 funnel tube for low pOll t'l lenses, 66, 69 hornal oculars, 80, 1]2, 166 macro-stage, 75 miflex camera, 48 mikrotars, 65, 66, 67 multiplicator back, 77, 78 optical bench, 76; 95 photomicrographic cameras, 5 I, 53,54,58,59 planars, 65 quartz slides and holder, 223 ultraphot, 55, 58, 62, 76 ultra-violet outfit, 220, 222 Zieler, 'W., on photomicrograph I with the Leica camera, I l()
Zeiss,