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Handbook of Microscopy

Marcel Locquin and Maurice L a n g e r o n


Translation edited by
Harold Hillman,
R e a d e r in P h y s i o l o g y , U n i v e r s i t y of S u r r e y

Butterworths
London Boston Durban Singapore Sydney Toronto Wellington
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English edition first published, 1983

Butterworth & Co (Publishers) Ltd, 1983

British Library Cataloguing in Publication Data

Locquin, Marcel
Handbook of microscopy.
1. Microscope and microscopy
I. Title I I . Langeron, Maurice
I I I . Manuel de microscopie. English
502.8 QH205.2

ISBN 0 - 4 0 8 - 1 0 6 7 9 - 4

This is the English translation of Manuel de Microscopie published in France


by Masson
Masson, Editeur, 1978

Typeset by Scribe Design, Gillingham, Kent


Printed by Cambridge University Press, Cambridge
Preface

T h e l a t e M a u r i c e L a n g e r o n arid I s h a r e d t w o often in a n a w k w a r d s i t u a t i o n . If his s y s t e m a t i c


c o m m o n passions, microscopy and mycology. a n d a n a t o m i c a l k n o w l e d g e is sufficient, h e will
T h e y s o o n b r o u g h t u s t o g e t h e r for, h a v i n g cor- p r o b a b l y find a n o t h e r s i m i l a r object, s t a r t i n g
r e s p o n d e d w i t h h i m j u s t before t h e S e c o n d f r o m w h i c h h e m i g h t c o n c e i v e of a n e w efficient
W o r l d W a r , I m e t h i m o n m y a r r i v a l in P a r i s m e t h o d of s t u d y . H i s l i t e r a t u r e s e a r c h m a y l e a d
a n d we soon discovered that our personalities h i m o n to difficult q u e s t i o n s : h o w to c h o o s e t h e
w e r e v e r y s i m i l a r . I n 1946, w e s t a r t e d a series of b e s t of s e v e r a l h u n d r e d fixatives, h o w to c h o o s e
weekly discussions on the then spectacular de- t h e b e s t s t a i n a m o n g t h o u s a n d s of w h i c h m a n y
v e l o p m e n t s of m i c r o s c o p y a n d its b i o l o g i c a l a r e k n o w n u n d e r s e v e r a l n a m e s , a n d h o w to
a p p l i c a t i o n s , p a r t i c u l a r l y in r e l a t i o n t o p a r a - r e m e m b e r t h e few a p p l i c a b l e t e c h n i q u e s from
sitology a n d m y c o l o g y . J u s t before t h e p u b l i c a - a m o n g t h e h u n d r e d s of t h o u s a n d s w h i c h h a v e
t i o n of t h e s e v e n t h e d i t i o n of his s t a n d a r d w o r k b e e n published, m a n y with variations. T h e en-
Prcis of Microscopy, w h i c h p r o v e d to b e his l a s t c y c l o p a e d i c c o l l e c t i o n s , like t h o s e of M c C l u n g
p u b l i c a t i o n , w e h a d a g r e e d o n t h e n e c e s s i t y to o r t h e Bolles L e e , will n o t h e l p h i m u n l e s s h e h a s
publish a completely revised eighth edition, a l r e a d y a c q u i r e d a g o o d g r o u n d i n g of h i s t o l o g y
r e c a s t in a n e w f o r m o n w h i c h w e h a d j o i n t l y a n d c y t o l o g y . S p e c i a l i s t b o o k s m a y confuse h i m
d e c i d e d . H o w e v e r , t h e d e a t h of M r L a n g e r o n b e c a u s e of t h e m u l t i p l i c i t y of o v e r l a p p i n g t e c h -
p r e v e n t e d u s from c o m p l e t i n g this n e w p r o j e c t . n i q u e s , o r d i s h e a r t e n h i m b y t h e o m i s s i o n of
I n 1957, w e h a d u n d e r t a k e n to w r i t e a n d e s s e n t i a l d e t a i l s . A n y logical t h r e a d m a y b e
p u b l i s h a Treatise of Microscopy in c o l l a b o r a t i o n s c a t t e r e d in different b o o k s , m o r e p h y s i c o -
w i t h A l b e r t P o l i c a r d a n d M a r c e l Bessis. T h i s c h e m i c a l t h a n b i o l o g i c a l , a n d h e will h a v e to
w a s n o t i n t e n d e d e i t h e r to r e p l a c e o r to follow i n t e g r a t e t h e different d i s c i p l i n e s , w i t h w h i c h h e
L a n g e r o n ' s Prcis, b e c a u s e it r e m a i n s i r r e p l a c e - m a y n o t b e a c q u a i n t e d , to b e a b l e to e x t r a c t t h e
a b l e as far a s t h e i n f o r m a t i o n c o n t a i n e d in it is m a i n ideas.
concerned. I u n d e r t o o k t h e t a s k of w r i t i n g this b o o k ,
B o t h of t h e s e b o o k s , p u b l i s h e d b y M a s s o n despite the justified pessimism shown by M r
a n d C o . , a r e n o w o u t of p r i n t a n d s i n c e light G a b e in 1968 c o n c e r n i n g t h e p o s s i b i l i t y of w r i t -
microscopy has m a d e great strides d u r i n g the i n g a n u p - t o - d a t e m a n u a l of m i c r o s c o p y . O p t i -
past twenty years, I started again on the project m a l l y , it s h o u l d c o m p r i s e , w i t h i n a m a n a g e a b l e
w e h a d o n c e d r a w n u p in t h e q u i e t i s o l a t i o n of l e n g t h , b o t h a logical c h o i c e of s t a i n i n g s y s t e m s
his l a b o r a t o r y in t h e r u e d e l ' E c o l e d e M d e c i n e , m e n t i o n e d earlier, a n d a concise a n d detailed
a n d I d e c i d e d to c o m p l e t e it, w i t h t h e v a l u a b l e a c c o u n t of o l d a n d n e w m e t h o d s a s s e s s e d criti-
c o l l a b o r a t i o n of o u r j o i n t e d i t o r , in t h e form of c a l l y . U n f o r t u n a t e l y , I h a d to d o it o n m y o w n ,
this n e w Handbook of Microscopy. s i n c e M a u r i c e L a n g e r o n is n o l o n g e r w i t h m e to
W h e n a m i c r o s c o p i s t finds a n e w object, h e is h e l p w i t h t h i s Handbook of Microscopy. I t is

vi Preface

p u b l i s h e d in E n g l i s h b y B u t t e r w o r t h s a n d in a n d following t h i s b y m o r e specific a d a p t a t i o n s .
F r a n c e b y M a s s o n , a n d is i n t e n d e d to c o m p l e - F i n a l l y , it is i n t e n d e d t o b e b r o a d l y b a s e d , since
m e n t t h e c l a s s i c b o o k b y M r G a b e , Histological the general charts, indispensable concepts and
Techniques, w h i c h g o e s m o r e d e e p l y i n t o h i s - b r i e f s y s t e m a t i c s u r v e y of m o s t of t h e b a s i c
t o c h e m i s t r y . I t d e a l s m a i n l y w i t h biological m e t h o d s u s e d in m o d e r n l i g h t m i c r o s c o p y a r e
a p p l i c a t i o n s of l i g h t m i c r o s c o p y , l e a v i n g q u a n - i n t e n d e d t o e n a b l e a n y s p e c i a l i s t to find in-
t i t a t i v e a n a l y s i s to m o r e s p e c i a l i z e d b o o k s s u c h f o r m a t i o n a b o u t t h e a p p l i c a t i o n of k n o w n t e c h -
as the one mentioned above. n i q u e s to a p a r t i c u l a r r e s e a r c h p r o j e c t a n d to t h e
T h e s e l e c t i o n is c r i t i c a l , s i n c e r e p e t i t i v e u s e s d e v e l o p m e n t of e n t i r e l y n e w t e c h n i q u e s .
h a v e been eliminated; the best reagents have S i n c e t h e F r e n c h e d i t i o n of this b o o k w a s
b e e n c h o s e n for r o u t i n e h i s t o l o g y i r r e s p e c t i v e of p u b l i s h e d b y M a s s o n in 1978, a b o u t 60 n e w
c u r r e n t p r a c t i c e , s u c h a s t h e u n j u s t i f i a b l e prefer- techniques have been added. In particular, a
e n c e of e t h a n o l o v e r a c e t o n e , of t o l u e n e o v e r l a r g e s e c t i o n h a s b e e n d e v o t e d to t h e s t u d y of
b e n z e n e a n d of t h e fixatives of H o l l a n d e o r t h e o r g a n i c m a t t e r in microfossils p r e s e r v e d in
Z e n k e r o v e r t h o s e of B o u i n a n d K o p s c h - R e g a u d r o c k s , in v i e w of its s t r a t i g r a p h i e i m p o r t a n c e in
for r o u t i n e u s e . T h e s e l e c t i o n s h a v e b e e n p r o g - m i n i n g a n d oil p r o s p e c t i n g .
ressive, starting with the o p t i m a l reagent or
t e c h n i q u e , d i s c u s s i n g t h e v a r i a n t s in g e n e r a l u s e Marcel V. Locquin
Acknowledgements

T h e c o l l e c t i o n a n d c o m p i l a t i o n of t h i s m a n u a l G r a n d ) ; t e c h n i q u e s of l i g h t m i c r o s c o p y a t W i l d
h a s b e e n l o n g a n d e x a c t i n g a n d h a d to b e C o . ; p h o t o m i c r o g r a p h y at the Socit L u m i r e ;
constantly u p d a t e d . T h e late M a u r i c e L a n g e r o n c i n e m a t o g r a p h y at the Collge de France with J .
d e v o t e d a t l e a s t o n e h o u r e v e r y d a y to t h i s w o r k . D r a g e s c o ; t e l e m i c r o s c o p y a t t h e T h o m s o n Soci-
A t p r e s e n t , s y s t e m a t i c a b s t r a c t i n g of t h e l e a d i n g ety a n d later with Philips; phase contrast a n d
a n a l y t i c j o u r n a l s in all r e l e v a n t b r a n c h e s of interference contrast microscopy at Wild Co.;
s c i e n c e m a k e s it p o s s i b l e to o b t a i n a fairly polarized light microscopy with Messrs. Cotton
c o m p l e t e c o l l e c t i o n of t h e l i t e r a t u r e . T h e m o s t a n d M a n i g a u l t at Bellevue; spodography at the
fruitful s o u r c e s a r e t h e c o m p r e h e n s i v e t e x t - H i s t o l o g y L a b o r a t o r y of t h e F a c u l t y of M e d i c i n e
books, colloquia or congresses a n d the major in L y o n (A. P o l i c a r d ) ; s p a r k s p e c t r o g r a p h y a t
m i c r o s c o p i c j o u r n a l s . T h e r e is n o r e c e n t w o r k in t h e F o r e n s i c L a b o r a t o r y w i t h Prof. S a n n i e ; diffe-
F r e n c h on light microscopy, unlike electron r e n t m e t h o d s of l i g h t i n g w i t h M e s s r s . C l a u d e
microscopy. Periodic and systematic enquiries P a z a n d Silva; s u r f a c e t r e a t m e n t s a n d intefer-
m a d e a t a r e p r e s e n t a t i v e s a m p l e of l a b o r a t o r i e s e n c e filters w i t h M e s s r s G A B ; flashlights a n d
d o i n g m i c r o s c o p y m a d e it p o s s i b l e to k e e p lasers with the O r t h o t r o n Co.; botany and
abreast with current techniques. Such enquiries m y c o l o g y a t t h e L a b o r a t o r y of C r y p t o g a m y of
m u s t obviously be supranational. F a r too m a n y t h e P a r i s M u s e u m (Prof. R . H e i m ) ; p a l y n o l o g y
p e o p l e h a v e h e l p e d u s in t h i s w a y to e n a b l e u s to a n d micropalaeontology at the Paris M u s e u m
t h a n k t h e m i n d i v i d u a l l y . I n d e e d , e v e n before laboratory (G. Deflandre), at the Micropalaeon-
t h e fifth e d i t i o n of his Prcis, M a u r i c e L a n g e r o n t o l o g y L a b o r a t o r y of t h e U n i v e r s i t y of P a r i s V I
h a d g i v e n u p t r y i n g to c h e c k h i m s e l f t h e v a l u e of with M r s T a u g o u r d e a u and M r Lashkar, and at
all t h e t e c h n i q u e s h e m e n t i o n e d . t h e L a b o r a t o r y of t h e E c o l e P r a t i q u e d e s H a u t e s
M a r c e l L o c q u i n insisted on being present Etudes with M r Taugourdeau; haematology
w h e n p r o c e d u r e s w e r e p e r f o r m e d in a s p e c i a l i s t a n d c y t o l o g y a t t h e L a b o r a t o r y of t h e B l o o d
l a b o r a t o r y w h e n e v e r h e w a s u n a b l e to c a r r y T r a n s f u s i o n C e n t r e w i t h M r Bessis; d o c u m e n t a -
t h e m o u t himself. H e p e r s o n a l l y p e r f o r m e d ex- tion with Phototechnique and with M r Cordon-
p e r i m e n t s o n t h e following s u b j e c t s a t t h e p l a c e s nier; m i c r o p h o t o m e t r y with Europelec Co.; pa-
i n d i c a t e d : t h e c h e m i s t r y of s t a i n s a n d fixatives r a s i t o l o g y a n d m y c o l o g y a t t h e I n s t i t u t e of
a t t h e I n s t i t u t e of C h e m i s t r y in L y o n a n d a t Parasitology with M r Langeron; protistology at
R h n e P o u l e n c ; light m i c r o s c o p y a t t h e I n s t i t u t e the Collge de F r a n c e with E. F a u r e Fremiet;
of O p t i c s u n d e r t h e g u i d a n c e of F r a n o n a n d a u t o m a t i o n in t h e a n a l y s i s of i m a g e s a t C E N -
Nomarski; colorimetry a n d electron microscopy F A R w i t h D r L e G . I w i s h to e x p r e s s m y v e r y
a t t h e P h y s i c s L a b o r a t o r y of t h e N a t i o n a l s i n c e r e t h a n k s to all of t h e m a n d to all t h e i r
M u s e u m of N a t u r a l H i s t o r y , P a r i s (Prof. Y . L e c o l l a b o r a t o r s w h o m a d e m y t a s k m u c h easier.

vit
viii Abbreviations

Abbreviations

Acidity or alkalinity pH Millimetre mm


Centimetre cm Minute min
Contrast Y Molar concentration M
Equal quantities aa Nanometre n m ( = rr
Gram g Normality
Hour h Optical density
Joule J Oxidation-reduction potential
Litre 1 Second s
Micrometre Temperature C o r K
Milligram mg Wavelength
3
Millilitre ml ( = c m )
1 Instruments and Techniques

1.1 Basic principles


Light and colours

T h e l u m i n o u s flux e m i t t e d b y a n o b j e c t c a n b e
a n a l y s e d b y different m e t h o d s . F o r t h i s p u r p o s e ,
t h e u n i t s u s e d a r e defined a s follows:

c = t h e s p e e d of light in a v a c u u m = 2 9 9 776
-1
km s
= t h e w a v e l e n g t h = c/v, w h e r e is t h e
frequency
4
= = m i c r o n = 1 0 ~ c m
7
n m = = m i l l i m i c r o n = 10~ c m
8
0.1 n m = A = a n g s t r m = 1 0 " c m
=
= t h e refractive i n d e x ; n^o t h e refractive
i n d e x of t h e y e l l o w D line of s o d i u m a t 20 C

Figure 1.1 s h o w s t h e r a n g e of r a d i a t i o n s w h i c h
e n c o m p a s s e s t h e o c t a v e of r a d i a t i o n s visible to
t h e eye, c o m p r i s i n g a b o u t o n e - s e v e n t i e t h of t h e
total k n o w n range. In microscopy, exploration
of t h e u l t r a v i o l e t a n d i n f r a r e d r e g i o n s e x t e n d s
t h e k n o w n r e g i o n s to b a r e l y t h r e e o c t a v e s {Figure
1.2).
Y o u n g a n d Fresnel, using interference a n d
diffraction, a t t h e b e g i n n i n g of t h e n i n e t e e n t h
F i g u r e 1.1. Diagram of radiations.
c e n t u r y , s h o w e d t h a t light v a r i e s p e r i o d i c a l l y in Leftwavelengths (m); middleconventional regions;
both time a n d space. T h u s , at a given point the rightenlarged diagram of the visible range
i n t e n s i t y of light r e a c h e s its m a x i m u m v a l u e and conventional regions of colour sensations
m a n y t i m e s p e r s e c o n d ; this is its f r e q u e n c y
(cycles p e r s e c o n d H z ) . I n s p a c e , t h e d i s t a n c e a b s o r p t i o n , a l t h o u g h it r e a d i l y a c c o u n t s for
s e p a r a t i n g t w o w a v e surfaces h a v i n g t h e s a m e p r o p a g a t i o n . A d i s c r e t e a s p e c t is a s s u m e d a
i n t e n s i t y is t h e w a v e l e n g t h . T h e s e t w o q u a n t i - q u a n t i z a t i o n of t h e e n e r g y t r a n s p o r t e d b y
ties a r e c o n n e c t e d b y t h e r e l a t i o n s h i p = cl v. lightto interpret these phenomena. Max
T h e p e r i o d i c n a t u r e of t h e w a v e d o e s n o t l e a d P l a n c k (1905) s h o w e d t h a t this q u a n t i z a t i o n
to a n e a s y i n t e r p r e t a t i o n of light e m i s s i o n a n d i n v o l v e s m u l t i p l e s of a n e l e m e n t a r y q u a n t i t y
/
2 Instruments and techniques

c a l l e d a q u a n t u m of e n e r g y : e = hv. E a c h T h e s p e c t r u m of a r a d i a t i o n s o u r c e m a y b e
q u a n t u m is p r e s e n t in t h e light w a v e in t h e form continuous or discontinuous. In the latter case
of a p h o t o n w h i c h c o n s t i t u t e s its e l e m e n t a r y w e s p e a k of a line s p e c t r u m ; s o m e s o u r c e s give
p a r t i c l e b u t w h i c h c a n n o t exist in a s t a t e of rest. rise to a m i x t u r e of b o t h . E x c i t a t i o n of a g a s s u c h
A c c o r d i n g to Y v e s le G r a n d , light c a n b e s a i d to a s s o d i u m o r m e r c u r y v a p o u r p r o d u c e s a line
c o n s i s t of a ' w a v e of d i s e m b o d i e d p a r t i c l e s ' . s p e c t r u m , while heating a tungsten filament
P o i n t s o u r c e s a r e seen by t h e o b s e r v e r o r b y r e s u l t s in a c o n t i n u o u s s p e c t r u m . A c o n t i n u o u s
t h e r e c e i v i n g a p e r t u r e of t h e i n s t r u m e n t u s e d a s s p e c t r u m is c h a r a c t e r i z e d b y its s p e c t r a l e n e r g y
a s m a l l a n g l e , n o t e x c e e d i n g a few a r c m i n u t e s . c u r v e , a n d a line s p e c t r u m b y t h e w a v e l e n g t h
T h e s m a l l e r t h e p o i n t s o u r c e , t h e m o r e t h e light a n d i n t e n s i t y of e a c h line.
is s a i d to b e ' c o h e r e n t ' ; t h e i m p o r t a n c e of this T h e eye c o n s t i t u t e s t h e b a s i c r a d i a t i o n r e c e p -
c o n c e p t will b e seen l a t e r . t o r , s i n c e n o o p t i c a l i n s t r u m e n t is c a p a b l e of
E x t e n d e d s o u r c e s m a y b e of low c o h e r e n c e , using an image by analysing photographed,
partially coherent or incoherent. T h e y are called d i s p l a y e d o r r e c o r d e d r e s u l t s d i r e c t l y o r in-
p r i m a r y s o u r c e s if t h e y a r e s e l f - l u m i n o u s a n d d i r e c t l y . P h o t o c e l l s o r p h o t o s e n s o r s b e l o n g to
s e c o n d a r y s o u r c e s if t h e y modify a p o r t i o n of t h e different c a t e g o r i e s , d e p e n d i n g o n t h e i r m o d e of
r a d i a t i o n t h e y receive. If a diffusing surface is f u n c t i o n i n g . T h e y a r e d e a l t w i t h in s p e c i a l i z e d
p l a c e d in front of a c o h e r e n t s o u r c e , a n i n c o h e - electronic a n d optical textbooks.
rent secondary source results. Therefore, such I n accordance with their chemical structure,
a n a t t a c h m e n t s h o u l d n o t b e u s e d if it is n e c e s - p h o t o s e n s i t i v e surfaces r e c o r d d e n s i t i e s in b l a c k
s a r y to p r e s e r v e t h e c o h e r e n c e of t h e s o u r c e , a n d w h i t e o r in c o l o u r . T h e s e r e c e p t o r s h a v e o n e
especially with p h a s e contrast, interference con- c h a r a c t e r i s t i c in c o m m o n : t h e y r e c o r d o n l y a m -
t r a s t , o r w i t h p o l a r i z e d light m i c r o s c o p y . Brief- p l i t u d e s o r d e g r e e s of g r e y in t h e i m a g e b u t
ly, it s h o u l d b e n o t e d t h a t a l a s e r b e a m h a s a n e v e r t h e p h a s e s . I n o p t i c s , a b l a c k b o d y is a
very marked coherence, which permits certain c o n c e p t of c o n s i d e r a b l e t h e o r e t i c a l i n t e r e s t . I n
s p e c i a l o p t i c a l p h e n o m e n a s u c h as h o l o g r a p h y . m i c r o s c o p y , it is useful o n l y a s a s t a n d a r d of
Radiance and brightness are two homologous r e f e r e n c e . G e n e r a l l y s p e a k i n g , all b o d i e s
c o n c e p t s , t h e first of w h i c h r e l a t e s to v o l u m e a n d observed under the microscope are non-black
t h e s e c o n d to surface. O n l y t h e r a d i a n c e of t h e bodies.
sky o r of a d i s c h a r g e in a g a s c a n b e of r e l e v a n c e At present, incandescent bulbs equipped with
to u s , w h i l e t h e b r i g h t n e s s of s o u r c e s is of t h e a f i l a m e n t h e a t e d b y t h e J o u l e effect a r e still t h e
g r e a t e s t i m p o r t a n c e in m i c r o s c o p y . I n d e e d it m o s t w i d e l y u s e d light s o u r c e s . T h e i r s p e c t r a l
c a n b e s h o w n t h a t , for a given a p e r t u r e of a n energy curve varies markedly with the current
o p t i c a l s y s t e m , t h e b r i g h t n e s s of t h e s o u r c e p a s s e d (see Figure 1.21). T h e i r c o l o u r t e m p e r a -
d e t e r m i n e s t h e l u m i n o s i t y of t h e i m a g e p r o - t u r e p r o v i d e s a c o n v e n i e n t m e a n s of m a r k i n g t h e
d u c e d by t h e m i c r o s c o p e . T h e r e f o r e , t h e d e - d o m i n a n t w a v e l e n g t h of t h e i r e n e r g y s p e c t r u m
v e l o p m e n t of m o r e powerful s o u r c e s h a s b e e n a (Figure 1.3).
factor in d e t e r m i n i n g a d v a n c e s in m i c r o s c o p y . R e t i n a l h e t e r o g e n e i t y limits t h e r e s o l v i n g

Short Near Medium


U.V. VISIBLE I.R. I.R. DRYING I.R.
>e -
(Photographic)

2 . 0 (Aim)

Figure 1.2. Relationship between the infrared, visible and ultraviolet regions
(According to M. Dribr, 1944)
Basic principles 3

RADIO PHOTONS X-RAYS

I.R. U.V.

I 1 1 h H 1 1 1 \ H 1 h (a)
1km 1m 1cm 1mm 1/im 1nm 0.1 nm 1pm

- 2 5 - 2 4 - 2 3 - 2 2 -21 -20 -19 - 1 8 -17 - 1 6 - 1 5 -14 -13


10 10 10 10 10 10 10 10 10 10 10 10 10 Ws

' ' '., . J


(6)
1 o 10 10^ 1 0 V
2 3 2 3
1 10 10 10 m 1 10 10 10 kV
2 3 F i g u r e 1.3. Physical data
1 10 10 10 1 10 M V (c) relating to waves and
particles utilized in
~~i 1 1 1I (d)
microscopy.
1M 10Onm 10nm 1 nm 0.1 nm 0 . 0 1 nm 1pm 0.1pm (a) wavelengths; (b) energy
quanta; (c) acceleration of
electrons; (d) wavelength
(e) associated with electrons;
"1 (e) wavelength associated
10nm 1 nm 0.1 nm 0 . 0 1 nm 1 prrr 0.1pm with protons

p o w e r of t h e e y e . I t is n o w k n o w n t h a t c o n e s a n d U l t r a v i o l e t light is h a r m f u l to t h e eye; f u r t h -
r o d s a r e c o n n e c t e d b y l a t e r a l fibres. T h i s m a k e s e r m o r e , it excites fluorescence a n d forms a
obsolete the previously held hypotheses p u t bluish 'halo' a r o u n d the images.
f o r w a r d to d e d u c e t h e t h e o r e t i c a l r e s o l v i n g p o w - T h e i n t e n s i t y of t h e light a t t h e r e t i n a r e g -
e r of t h e eye from t h e i r d i m e n s i o n s . F u r t h - u l a t e s t h e d i a m e t e r of t h e p u p i l reflexly. I n t h e
e r m o r e , t h e eye oscillates c o n t i n u o u s l y a b o u t a m i c r o s c o p e t h i s reflex h a s n o effect o n t h e flux
m e a n p o s i t i o n a n d e x p l o r e s m o r e t h a n it fixes. e n t e r i n g t h e e y e , s i n c e t h e field visible a t t h e
T h e eye h a s a c o m p l e x s p a t i o - t e m p o r a l m e m - eyepiece has generally a m u c h smaller diameter
ory. It establishes an a p p a r e n t continuity w h e n a t h i g h m a g n i f i c a t i o n . T h i s e x p l a i n s w h y t h e eye
d i s c o n t i n u o u s i m a g e s follow o n e a n o t h e r a s t h e y is easily d a z z l e d b y i n t e n s e light. F u r t h e r m o r e ,
d o in c i n e m a t o g r a p h y . A n a p p a r e n t c o n t i n u i t y t h e s e n s i t i v i t y of t h e r e t i n a c h a n g e s v e r y r a p i d l y
of o u r c o n s c i o u s n e s s m a s k s t h e i m a g e of d i s c o n - w i t h i n c r e a s i n g d i s t a n c e of t h e i n c i d e n t b e a m of
t i n u i t i e s in o u r c o n s c i o u s t h o u g h t a p r o c e s s l i g h t f r o m t h e a x i s of t h e eye; t h i s is t h e P u r k i n j e
w h i c h t a k e s p l a c e in t h e fully a w a k e s t a t e . effect. T h e eye b e h a v e s as if it c o n s i s t s of t w o
T h e v i s u a l field of t h e eye is a l w a y s g r e a t e r r e c e p t o r s f u n c t i o n i n g differently w i t h r e g a r d s to
t h a n t h a t of t h e i n s t r u m e n t i n t o w h i c h t h e eye is s e n s i t i v i t y to w a v e l e n g t h in full d a y l i g h t a n d in
l o o k i n g . T h e o p e r a t o r m u s t b e t r a i n e d to u s e d i m light.
p e r i p h e r a l vision for a r a p i d e x p l o r a t i o n of w h a t M o s t of t h e o b j e c t s e x a m i n e d u n d e r t h e
h e sees. H e m u s t b e a b l e to a v o i d c e n t r a l vision microscope are either naturally coloured or dyed
c o n s c i o u s l y , in f a v o u r of p e r i p h e r a l v i s i o n , w i t h - artificially. T h e r e f o r e , it is e s s e n t i a l to b e a r in
o u t m o v i n g his e y e s . T h i s is i n d i s p e n s a b l e for m i n d t h e following p r i n c i p l e s of c o l o u r m e a s u r e -
a c q u i r i n g t h e t e c h n i q u e of r a p i d e x p l o r a t i o n m e n t , to m a k e t h e b e s t u s e of a n i n s t r u m e n t .
z o n e b y z o n e , of w h i c h h i g h s p e e d r e a d i n g is a T h e c o l o u r w h i t e is o n l y w h i t e w i t h reference t o
particular case. a s t a n d a r d . T h i s is a set s t a n d a r d : it is d a y l i g h t
T h e l i m i t of visibility v a r i e s b e t w e e n o b s e r - a t 5 2 0 0 K ; it is k n o w n t h a t ' c o l o u r t e m p e r a t u r e '
v e r s a n d d e p e n d s u p o n t h e i n t e n s i t y of i l l u m i n a - v a r i e s c o n s i d e r a b l y d u r i n g t h e c o u r s e of a d a y
t i o n . T h e m a x i m u m visibility shifts from y e l l o w a n d from m o n t h to m o n t h . A m o n o c h r o m a t i c
to g r e e n w h e n t h e i l l u m i n a t i o n is d i m i n i s h e d . r a y is d e t e r m i n e d c o m p l e t e l y b y its w a v e l e n g t h
T h e i m a g e a t t h e e y e p i e c e of a m i c r o s c o p e is n o t a n d its flux. A c o l o u r e d r a y is defined c o m p l e t e -
a n a l o g o u s to a ' h o l e of l i g h t ' a t t h e b o t t o m of a ly b y its flux a n d b y t w o a d d i t i o n a l v a r i a b l e s : a
b l a c k t u n n e l . I n o r d e r to o b t a i n g r e a t e r s h a r p - w h i t e b a c k g r o u n d a n d a m o n o c h r o m a t i c colour.
n e s s a s s o c i a t e d w i t h g r e a t e r c o m f o r t , it w o u l d b e I n p r a c t i c e , p r e f e r e n c e is g i v e n to t h e s y n t h e s i s
n e c e s s a r y to h a v e a w e a k a u x i l i a r y i l l u m i n a t i o n of c o l o u r s b y t h e a d d i t i o n of t h r e e v a r i a b l e
a t t h e p e r i p h e r y of t h e field. T h i s h a s n o t y e t c o l o u r s ; t h i s is t h e t r i c h r o m e s y n t h e s i s c u r r e n t l y
b e e n tested in m i c r o s c o p y . u s e d in p h o t o g r a p h y a n d p r i n t i n g . H o w e v e r , in
4 Instruments and techniques

t h i s c a s e , t h e c o l o u r e d lights m u s t c o v e r b r o a d a s s i m i l a t e d o n a filter. If t h e y a r e m u l t i c o l o u r e d ,
b a n d s a n d not be monochromatic. a n u n d e r s t a n d i n g of w h a t is h a p p e n i n g c a n b e
E v e r y c o l o u r h a s its c o m p l e m e n t a r y c o l o u r gained by having previously separated out the
w h i c h , w h e n a d d e d to it, p r o d u c e s w h i t e ; for t r i c h r o m a t i c f a c t o r s , if n e c e s s a r y , b y s p e c t r o s -
e x a m p l e , yellow is c o m p l e m e n t a r y to b l u e - copy.
violet; t h e s e a r e all c o l o u r s of t h e s p e c t r u m , M o s t c o l o u r e d s o l u t i o n s follow B e e r ' s l a w , i.e.
e x c e p t for t h e c o m p l e m e n t of g r e e n w h i c h is t h e i r a b s o r p t i o n is p r o p o r t i o n a l to t h e c o n c e n -
p u r p l e , t h e o n l y p u r e c o l o u r n o t r e p r e s e n t e d in tration a n d the thickness traversed. T h e consti-
t h e s p e c t r u m . If t h e p u r e c o l o u r s a r e a r r a n g e d in t u e n t s of a m i x t u r e of d y e s in g e n e r a l also follow
a circle, t h e c o m p l e m e n t a r y c o l o u r s a r e d i a m e t - t h i s l a w if t h e y d o n o t r e a c t w i t h o n e a n o t h e r . I n
rically o p p o s i t e e a c h o t h e r . T h e t w o t r i a d s p o s s i - t h e c a s e of m i c r o s c o p y u s i n g a n e y e p i e c e , as
b l e for t r i c h r o m e s y n t h e s i s a r e f o u n d a t t h e o p p o s e d to p h o t o g r a p h i c m i c r o s c o p y , t h e o v e r -
a p i c e s of a n e q u i l a t e r a l t r i a n g l e . all a m o u n t of light w h i c h r e a c h e s t h e eye from
I n m i c r o s c o p y , five c a t e g o r i e s of c o l o u r s c a n t h e s o u r c e after p a s s a g e t h r o u g h filters a n d t h e
be distinguished: optical observation system must be taken into
c o n s i d e r a t i o n . I n a d d i t i o n , o n e h a s to t a k e i n t o
(a) t h e c o l o u r s f o r m e d b y selective a b s o r p t i o n a c c o u n t t h e p a s s a g e of light t h r o u g h t h e m e d i a
of a p i g m e n t ; of t h e eye. I t m u s t n o t b e f o r g o t t e n t h a t s o m e
(b) t h e c o l o u r s f o r m e d b y a b s o r p t i o n - r e f l e c t i o n ; filters c h a n g e c o l o u r a s a r e s u l t of a g e i n g . I n
(c) t h e c o l o u r s f o r m e d b y i n t e r f e r e n c e in n a t u - p a r t i c u l a r , t h e a q u e o u s a n d v i t r e o u s fluids yel-
r a l light; l o w w i t h a g e , w h i c h i n c r e a s e s t h e p e r c e p t i o n of
(d) t h e c o l o u r s f o r m e d b y d i f f u s i o n - d i s p e r s a l ; t h e w h i t e c o l o u r u s e d as reference.
(e) t h e c o l o u r s f o r m e d b y i n t e r f e r e n c e in p o l a - T h i s m e a n s t h a t in p r a c t i c e w e c a n n o t d o
r i z e d light. w i t h o u t reference colours. T h e s e are supplied by
t h e ' c o l o u r c o d e s ' . O n l y Chromotaxia b y M .
T h e eye is i n c a p a b l e of d i f f e r e n t i a t i n g b e t w e e n L o c q u i n , P a r i s (1965) allows c o m p a r i s o n b y
t h e s e v a r i o u s m e t h o d s of f o r m a t i o n in t h e s a m e t r a n s m i s s i o n . I n 1975, t h e s a m e a u t h o r p u b -
w a y t h a t it is i n c a p a b l e of r e c o g n i z i n g t h e l i s h e d a Guide to Natural Colours followed b y a
c o m p o s i t i o n of t w o different m i x t u r e s if t h e y table showing the correspondence between the
give t h e s a m e v i s u a l c o l o u r i m p r e s s i o n . c o l o u r s g i v e n in this g u i d e w i t h t h e b a s i c w o r k s
I n c o l o r i m e t r y a so-called X Y Z s y s t e m h a s of S a c c a r d o a n d R i d g w a y , a n d w i t h t h e M u n s e l l
b e e n a d o p t e d i n t e r n a t i o n a l l y , in w h i c h t h e notation. T h e latter work only enables compari-
t r i c h r o m a t i c c o o r d i n a t e s of all t h e visible col- s o n w i t h c o l o u r s e x a m i n e d b y reflection.
o u r s h a v e b e e n t r a n s f e r r e d to t h e i n s i d e of a Tonality, saturation and value are the
t r i a n g l e . T h u s , t h e c o l o u r e d filters c u r r e n t l y p s y c h o l o g i c a l v a r i a b l e s w h i c h c o r r e s p o n d to t h e
a v a i l a b l e a r e all r e p r e s e n t e d w i t h i n s u c h a p h y s i c a l q u a n t i t i e s of d o m i n a n t w a v e l e n g t h ,
t r i a n g l e b y m e a n s of a p o i n t a n d its t h r e e clarity a n d luminosity. O n l y the former vari-
c o o r d i n a t e s . T h e c o l o u r t e m p e r a t u r e s of t h e a b l e s a r e t a k e n i n t o a c c o u n t in p r a c t i c e , a n d
s o u r c e s lie o n a p a r a b o l a referred to a s t h e b l a c k t h e y s e r v e as t h e b a s i s of t h e m o s t u n i v e r s a l l y
body locus. a d o p t e d s y s t e m of c o l o u r n o t a t i o n .
W h e n a c o l o u r e d filter is i n t e r p o s e d b e t w e e n a T o n a l i t y is r e l a t e d to t h e d o m i n a n t p u r e
s o u r c e a n d t h e eye, t h e t r a n s m i t t e d light is c o l o u r , s a t u r a t i o n to its d e g r e e of p u r i t y o r to t h e
m o d i f i e d . If this filter is n e u t r a l , t h a t is if it r e c i p r o c a l v a l u e of w h i t e in t h e c o l o u r , a n d t h e
a b s o r b s all w a v e l e n g t h s e q u a l l y , t h e n t h e o p t i c a l v a l u e to t h e p e r c e n t a g e of g r e y in t h e c o l o u r .
d e n s i t i e s a d d t o g e t h e r w h e n t h e filters a r e s u p e r - H o w e v e r , it m u s t b e a d m i t t e d t h a t so far t h e r e
i m p o s e d ; if t w o filters a r e c o m p l e m e n t a r y t h e i r is n o s a t i s f a c t o r y t h e o r y of c o l o u r vision c a p a b l e
s u p e r i m p o s i t i o n s t o p s all light; w i t h o t h e r filters, of i n t e g r a t i n g all t h e k n o w n p h e n o m e n a , b u t
results m a y vary considerably, d e p e n d i n g on t h i s will n o t b e d i s c u s s e d f u r t h e r h e r e .
w h e t h e r t h e s o u r c e h a s a line s p e c t r u m o r a
continuous spectrum. In practice, a graphic
Image formation
s u p e r i m p o s i t i o n of t h e filter c u r v e s w i t h t h e
s p e c t r a l d i s t r i b u t i o n c u r v e of t h e s o u r c e m a k e s it O p t i c a l treatises usually begin with an explana-
p o s s i b l e to p r e d i c t t h e r e s u l t . W h e n t h e d e t a i l s t i o n of a s i m p l e m a g n i f y i n g l e n s , t h e n a c o m -
of t h e o b j e c t a r e of o n e c o l o u r , t h e y c a n b e p o u n d a p p a r a t u s , simulating the objective-
Basic principles 5

Projection screen

S l i d e Projector Q
-////- W i t h o u t eyepiece

A"

Eyepiece

Illuminating optics

I m a g e of t h e
lamp filament First-stage magnification
J Eyepiece
Microscope

Intermediate image

Figure 1.4. Path of light rays in a slide projector and in a microscope (Leitz-Wetzlar)

e y e p i e c e r e l a t i o n s h i p of t h e m i c r o s c o p e b y v e l o p e d from s t a n d a r d s e s t a b l i s h e d b y t h e R o y a l
m e a n s of t w o single l e n s e s . T h i s a r r a n g e m e n t , M i c r o s c o p i c a l Society in G r e a t B r i t a i n , e s p e c i a l -
w h i c h is n o t v e r y c o n v i n c i n g to t h o s e w h o h a v e ly in r e s p e c t of t h e s c r e w t h r e a d to m o u n t t h e
n o t s t u d i e d p h y s i c s , h a s b e e n d e s c r i b e d so m a n y objective on the revolving nosepiece. T h e length
t i m e s t h a t it will n o t b e r e p e a t e d h e r e . H o w e v e r , of t h e o b j e c t i v e s v a r i e s a b o u t a m e a n of 35 m m
it is w i d e l y u s e d a t p r e s e n t . I n o u r o p i n i o n it is f r o m o n e d e s i g n e r to a n o t h e r .
closer to r e a l i t y t h a n t h e m o d e l of t h e m i c r o - W h e n the designer incorporates corrected
scope lens, w h e t h e r we are dealing with the o b j e c t i v e s in t h e m i c r o s c o p e to p r o d u c e a n
c o m m o n p h o t o g r a p h i c slide p r o j e c t o r o r w i t h i m a g e a t infinity, t h i s i m a g e is b r o u g h t to t h e
t h e e n l a r g e r u s e d for p h o t o g r a p h i c n e g a t i v e s . I n
Figure 1.4, t h e d i r e c t i o n of t h e r a y s of light is
h o r i z o n t a l a t t h e t o p in a p r o j e c t o r a n d a t t h e
b o t t o m in a m i c r o s c o p e . T h e s e t w o d i a g r a m s d o Mechanical
length of
not require long explanations. Finally, the read- tube
e r is r e f e r r e d t o t r e a t i s e s d e a l i n g w i t h t h e o p t i c s
a n d m e c h a n i c s of t h e m i c r o s c o p e for m o r e p r e -
cise d e t a i l s .

Mechanical conventions
S o m e m e c h a n i c a l p a r a m e t e r s r e l a t i n g to m i c r o -
s c o p e s h a v e b e e n s t a n d a r d i z e d for s o m e t i m e .
T h e t u b e l e n g t h of m o n o c u l a r i n s t r u m e n t s is
F i g u r e 1.5. Mechanical standardization of the
u s u a l l y 160 m m , b u t in L e i t z i n s t r u m e n t s it is microscope (Wild-Heerbrugg)
170 m m a n d in i n v e r t e d m i c r o s c o p e s it is g e n e r -
ally 250 m m . T h i s l e n g t h is m e a s u r e d b e t w e e n observation end point by a lens, a n d intermedi-
t h e o b j e c t i v e a t rest a n d t h e e y e p i e c e a t rest. a r y d e v i c e s c a n b e i n t e r p o s e d d i r e c t l y in front of
W h e n a n a c c e s s o r y is i n t r o d u c e d w h i c h a l t e r s this lens. T h i s obviously represents the best
this length, an optical system m u s t be incorpo- s o l u t i o n . I n p a r t i c u l a r , it a l l o w s t h e i n t r o d u c t i o n
r a t e d to c o m p e n s a t e for this c h a n g e . T h i s is of a n o p t i c a l d e v i c e specially d e s i g n e d to r e a l i g n
n e a r l y a l w a y s t h e c a s e for b i n o c u l a r t u b e s , d r a w t h e focal p l a n e of t h e o b j e c t i v e , t h e r e b y m a k i n g
t u b e s a n d a l m o s t all a c c e s s o r i e s b e t w e e n t h e v a r i o u s o p e r a t i o n s m o r e c o n v e n i e n t , e.g. t h e
s t a n d a n d t h e o b s e r v a t i o n d e v i c e (Figure 1.5). i n t r o d u c t i o n of p h a s e p l a t e s a n d v a r i o u s c o m -
T h e s t a n d a r d i z a t i o n of o b j e c t i v e s w a s d e - pensators.
6 Instruments and techniques

Aberrations s p h e r i c a l c a p . T h e c e n t r e a n d t h e e d g e of
the image cannot simultaneously be
A b e r r a t i o n s a r e called t h e c o r r e c t i o n defects of
b r o u g h t i n t o focus. T h i s defect b e c o m e s
an optical system. In microscopy there are three
m o r e a w k w a r d as t h e field b e c o m e s l a r g e r .
i m p o r t a n t t y p e s of a b e r r a t i o n :
I t is m u c h m o r e o b v i o u s in p h o t o m i c r o g r a -
(1) S p h e r i c a l a b e r r a t i o n , w h i c h m u s t n o t b e p h y t h a n in o b s e r v a t i o n b y eye, b e c a u s e t h e
c o n f u s e d w i t h field c u r v a t u r e . S p h e r i c a l eye f r e q u e n t l y refocuses, a n d also d u r i n g
a b e r r a t i o n is c a u s e d b y t h e p r i s m effect of a observation one frequently m a n i p u l a t e s the
s i n g l e lens w h i c h forms a b a n d of r a y s from fine focus, w h i c h r e s u l t s in a c o n t i n u o u s
a point source r a t h e r t h a n a point i m a g e as c o m b i n a t i o n of s e v e r a l p l a n e s of t h e object.
expected. T h i s can be corrected by
A f o u r t h a b e r r a t i o n t h e c o m a w h i c h is less
a p p r o p r i a t e design or modifications, b o n d -
a p p a r e n t , is c a u s e d b y c e n t r i n g defects of t h e
i n g t o g e t h e r t w o o r t h r e e r e f r a c t i n g lenses
different c o n s t i t u e n t p a r t s of t h e o b j e c t i v e . By
w i t h different d i s p e r s i v e p o w e r s {Figure 1.6).
examining the small details against a black
(2) C h r o m a t i c a b e r r a t i o n , d u e to t h e v a r i a t i o n
b a c k g r o u n d , one can check w h e t h e r the centring
of focal d i s t a n c e w i t h w a v e l e n g t h . T h i s c a n
is perfectly s y m m e t r i c a l ; t h e i m a g e from a l u m i -
b e c o r r e c t e d in t h e s a m e m a n n e r (Figure
nous point on a black background shows a
1.7).
c o m e t - l i k e l a t e r a l tail if it is n o t .
(3) F i e l d c u r v a t u r e , d u e to t h e fact t h a t g e n e r a l -
ly t h e i m a g e g i v e n b y t h e o b j e c t i v e , f o r m e d
from a p l a n e o b j e c t , is in t h e f o r m of a Depth of field
Image of object T h e t e r m d e p t h of field refers to t h e d i s t a n c e
(air bubbles in w a t e r )
a l o n g t h e o p t i c a l axis w h i c h s e p a r a t e s t h e ex-
t r e m e p a r t s of t h e o b j e c t w h i c h a r e o b s e r v e d
sharply without mechanically changing the

focus. D u r i n g o b s e r v a t i o n w i t h t h e eye, t h e
o p e r a t o r c o m p e n s a t e s for a s h a l l o w field d e p t h
b y m o v i n g t h e fine focus b a c k a n d forth o n
a - e i t h e r s i d e of t h e m e a n p l a n e of o b s e r v a t i o n .
P h o t o g r a p h y a c h i e v e s t h e s a m e effect b y u s i n g a
b-
s u c c e s s i v e s u p e r i m p r e s s i o n t e c h n i q u e , as d e -
c
scribed later.
T o i n c r e a s e t h e field d e p t h , t h e l o w e s t m a g n i -
fication by the objective compatible with the
(a) (b) (c)
desired resolution and the highest magnification
Figure 1.6. Checking the correction of the by the eyepiece are chosen. In p h o t o g r a p h y the
spherical aberration of a microscope. Toptwo print m a y be enlarged, the negative being taken
defocalized images
w i t h b o t h o b j e c t i v e a n d e y e p i e c e of t h e l o w e s t
possible power.

Flatness of field
A field is flat if t h e i m a g e is d i s t i n c t , t h i n a n d flat
u p to the edge. T h e objective-eyepiece combina-
t i o n gives this r e s u l t . T h e r e f o r e , it s h o u l d n o t b e
altered subsequently.

T h e m i c r o s c o p i c image in wave optics


S o far o n l y t h e g e o m e t r i c a l a s p e c t of t h e p a t h of
light rays t h r o u g h the microscope has been
Figure 1.7. Chromatic aberrations of a simple lens.
considered. T h i s explains image formation at
Continuous linered; dotted lineblue. Going out of
focus on both sides of F, we obtain a blue image on the v e r y low p o w e r m a g n i f i c a t i o n , b u t a t h i g h m a g -
far side and a red one on the near side nification t h e diffraction c a u s e d b y t h e s t r u c t u r e
Basic principles 7

of t h e o b j e c t b e c o m e s v e r y i m p o r t a n t a n d m u s t e n a b l e a n i m a g e of t h e o b j e c t to a p p e a r . Figures
be taken into consideration. Abbe, the true i.<9and 7.9, i l l u s t r a t e w h a t h a p p e n s in t h e m i c r o -
f o u n d e r of m o d e r n l i g h t m i c r o s c o p y , first scope objective.
pointed o u t this necessity. A n object-micrometer
is sufficient to p e r f o r m t h e following s i m p l e
e x p e r i m e n t ; t h e o b j e c t - m i c r o m e t e r c o n s i s t s of a
g r i d of b l a c k lines o n a w h i t e b a c k g r o u n d . O n e
o b s e r v e s t h e e n d a t w h i c h t h e lines a p p e a r closer
t o g e t h e r u n d e r h i g h m a g n i f i c a t i o n a n d after fine
focusing, a n d t h e n r a i s e s t h e e y e p i e c e to v i e w
t h e focal p l a n e b e h i n d t h e o b j e c t i v e t h r o u g h a n
e y e p i e c e t e l e s c o p e of t h e t y p e u s e d for c e n t r i n g
phase plates. O n e then stops d o w n the aperture
d i a p h r a g m of t h e c o n d e n s e r a s far as p o s s i b l e ,
w h i c h p r o d u c e s a series of s p o t s w i t h c o l o u r e d
e d g e s a n d w h i t e c e n t r e s w h i c h b e c o m e visible in
t h e focal p l a n e . T h e l a t t e r m a x i m u m is c a l l e d 0
a n d t h e m a x i m a of t h e c o l o u r e d s p o t s a r e of t h e
o r d e r of 1, 2, 3 , e t c . , p r o g r e s s i v e l y w i t h i n c r e a s -
i n g d i s t a n c e from t h e a x i s . W h e n t h e o b j e c t is
r a i s e d , it is c o n f i r m e d t h a t o n l y z e r o o r d e r exists.
W i t h t h e o b j e c t in p o s i t i o n , t h e m a g n i f i c a t i o n is
r e d u c e d b y m e a n s of t h e o b j e c t i v e . W i t h a
sufficiently low p o w e r o b j e c t i v e , t h e r e is a m a x -
i m u m of z e r o o r d e r , a n d w i t h t h e e y e p i e c e r e s e t F i g u r e 1.9. Ringed conoscopic image next to the schematic
t h e o b j e c t b e c o m e s c o m p l e t e l y invisible a t t h i s orthoscopic image of the diatom serving as the test
i n s t a n t , i r r e s p e c t i v e of t h e p o w e r of t h e e y e p i e c e . microorganism, Pleurosigma angulatum. I, central
illumination; I I - V I , oblique illumination. It can be seen
I t is sufficient to s u b s t i t u t e a n o b j e c t i v e of
that the complete image consists of incomplete, oriented
a d e q u a t e n u m e r i c a l a p e r t u r e to a d m i t a t l e a s t images which give rise to an impression of false structures
t w o diffraction m a x i m a in t h e o r d e r of 1 to (Langeron)

Specimen

Objective
+m -m
Aperture
Focal p l a n e w i t h - ' diaphragm
diffraction m a x i m a

Wavelength
of t h e light

10

r Image r

j w i t h r e p r o d u c t i o n of i n t e n s i t i e s i F i g u r e 1.8. Influence of the aperture of an


sina> objective on the resolution and on the
sira>2A
d accuracy of the image. O n the right there
Resolving p o w e r = ~ are two secondary maxima and on the left
sin-a five
8 Instruments and techniques

I n o p t i c s it c a n b e s h o w n t h a t if is t h e U s e f u l magnification
greatest angle that a ray can make with the It h a s been shown t h a t a n objective which
o p t i c a l axis so t h a t it m a y still e n t e r t h e o b j e c - resolves t w o p o i n t s m u s t p o s s e s s a n u m e r i c a l
tive a p e r t u r e , t h e s m a l l e s t d i s t a n c e seen s e p a r - a p e r t u r e , sin a , w h i c h is sufficient t o a d m i t a t
a t e l y t h r o u g h t h e o b j e c t i v e is d e t e r m i n e d b y t h e l e a s t a diffraction m a x i m u m of t h e o r d e r of 1.
equation H o w e v e r , t h e observer c a n only perceive two
p o i n t s if t h e e y e is a b l e t o s e p a r a t e t h e m . T h e
0.6 a n g u l a r p o w e r of s e p a r a t i o n of t h e e y e is 2 ' ,
dn =
w h i c h c o r r e s p o n d s t o 0.15 m m s e e n a t a d i s t a n c e
sin
of 2 5 0 m m . T h e r e f o r e , it is e s s e n t i a l t h a t t h e
w h e r e is t h e w a v e l e n g t h of t h e light u s e d , s i n eyepiece enlarges t h e i m a g e given by the objec-
t h e n u m e r i c a l a p e r t u r e of t h e d r y o b j e c t i v e u s e d , tive in s u c h a w a y t h a t t w o d e t a i l s of t h e object
a n d t h e refractive i n d e x of t h e i m m e r s i o n a p p e a r s e p a r a t e d b y a t least this v a l u e . P r a c t i c a l
m e d i u m for h i g h p o w e r o b j e c t i v e s (n = 1 b y e x p e r i e n c e s h o w s t h a t for o b j e c t s of m e d i u m
d e f i n i t i o n for d r y objectives) (Figure 1.10). c o n t r a s t it is n e c e s s a r y to i n c r e a s e t h e n u m e r i c a l
a p e r t u r e of t h e o b j e c t i v e a b o u t 800-fold. I n all
e v e n t s , t h i s factor s h o u l d b e b e l o w 5 0 0 t i m e s t h e
n u m e r i c a l a p e r t u r e since o t h e r w i s e t h e r e is a
d a n g e r of losing d e t a i l s , b u t it is p r e f e r a b l e t o
a d o p t a f a c t o r of 1000 (Figures 1.12 a n d 1.13).
I n p h o t o g r a p h y a n i m a g e is f o r m e d b y p a r t i -
cles in a n e m u l s i o n , t h e p r o c e s s b e i n g a c c o m p a -
n i e d b y s o m e diffusion of light w i t h i n t h e sensi-
tive l a y e r . I n p r a c t i c e , a diffusion circle of t h e
o r d e r of 1/60 m a y b e a s s u m e d . T h i s r e s u l t s in a
magnification equal to 80 times the numerical
a p e r t u r e o n t h e film, w i t h t h e p o s s i b i l i t y of a
f u r t h e r 10-fold e n l a r g e m e n t so a s t o o b t a i n t h e
y
s a m e r e s o l u t i o n in t h e i m a g e o n t h e p a p e r a s in
t h e i m a g e p r o d u c e d in t h e e y e . H o w e v e r , a s
F i g u r e 1.10. Limit of resolution according to Rayleigh.
w o r k i n g t o t h e limits s o m e t i m e s c a u s e s e y e
This corresponds to the image of the Airy disc, d
f a t i g u e , t h e o p e r a t o r is s t r o n g l y a d v i s e d t o e x -
tend the magnification u p to 1600-1800 times
the numerical aperture. W h e n the magnified
Objective i m a g e s a r e t o b e v i e w e d collectively for a g r e a t e r
distance, magnification c a n be increased u p to
3 6 0 0 t i m e s t h e n u m e r i c a l a p e r t u r e (Figures 1.12
a n d 1.13). W i t h i n c r e a s i n g e y e p i e c e m a g n i f i c a -
t i o n , t h e i n t e r n a l defects b e c o m e p r o g r e s s i v e l y
m o r e t r o u b l e s o m e , especially for o l d e r p e r s o n s .
Several remedies have been proposed to over-
F i g u r e 1.11. Aperture of an objective. 2n sin a, where a
is half the angle of the aperture. If the objective is used in c o m e t h i s . T h e s i m p l e s t of t h e s e is u n d o u b t e d l y
air, = 1; in the case of an immersion objective, is o b s e r v a t i o n of a n i m a g e p r o j e c t e d o n a n o n -
greater than 1.5 p a r t i c u l a t e s c r e e n b y reflection o r o n a s t e p p e d
F r e s n e l lens b y t r a n s m i s s i o n , t a k i n g c a r e t o
I n p r a c t i c e , it is r a r e to w o r k a t full c o n d e n s e r m a k e t h e p o s i t i o n of t h e e y e c o i n c i d e w i t h its
d i a p h r a g m a p e r t u r e , especially b e c a u s e i m m e r - focus.
sion of t h e objectives also r e q u i r e s i m m e r s i o n of
t h e c o n d e n s e r s (Figure 1.11). T h e e q u a t i o n m u s t H o m o g e n e o u s immersion o f objectives
t a k e i n t o a c c o u n t this l i m i t a t i o n b y r e p l a c i n g T h e t h e o r e t i c a l m a x i m u m n u m e r i c a l a p e r t u r e of
sin b y o b j e c t i v e s in a i r is 1.0. T h e p r a c t i c a l l i m i t lies a t
0 . 9 5 , b u t few objectives a r e c a p a b l e of a t t a i n i n g
sin objective sin 0 c o d
n e n sr e
t h i s v a l u e . A m i c i w a s t h e first t o d e s i g n o b j e c -
2 tives h a v i n g t h e i r front i m m e r s e d in a n oil w i t h
Basic principles 9
!
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 . 8 0 . 9 | 1.0 1.1 1.2 1.3 1.4 1.5 1.6

tf I I
Aperture number I _L.

Focal Magni
(length) ficatior
160mm tube

'32 mm 5x
5x 20x
16 m m 10x r
5 x 1 Ox 20x 30x
8 mm 20x -1
1 1
CO < 5x 10x 15x 20x 25x 30x 35x
4 mm 40x I 1
5x 10x 15x

Obje<

3 mm 20x 25x
60x I
5x 10x 15x 20x
^ 2 mm 90x
5x 10x 15x 20x

R e s o l u t i o n () 2.75 1.39 0 . 9 2 . 6 9 0 . 5 5 0 . 4 6 0 . 4 0 0 . 3 5 5 5 0 . 3 0 5 0 . 2 7 5 0 . 2 5 0 . 2 3 0 . 2 1 1 0 . 2 0 0 . 1 8
R=X/2 0 . N . X=0.55jLtm
L
I I I I I I I I I I I I I I I
Overall m a g n i f i c a t i o n
( D i a m e t e r s ) = 1 0 0 0 "
n " I
100 500 1000 1500 1800

F i g u r e 1.12. Usable combinations of eyepieces (horizontally) and objectives (vertically). The vertical arrows join optimum
combinations for a conventional tube length of 160 mm. Broken linesachromatic systems; continuous linesapochromatic
systems
t h e s a m e r e f r a c t i v e i n d e x a s t h e glass of w h i c h
t h e o b j e c t i v e w a s m a d e . T h i s offers n u m e r o u s
a d v a n t a g e s : s u p p r e s s i o n of all reflections o n t h e
l a m i n a a n d o n t h e s u r f a c e of t h e o b j e c t i v e a n d ,
a b o v e a l l , a n i n c r e a s e of t h e n u m e r i c a l a p e r t u r e
t o 1.40. I m m e r s i o n o b j e c t i v e s h a v e b e e n p r o -
d u c e d u s i n g l i q u i d s w i t h m u c h h i g h e r refractive
indices to a d v a n c e further in this direction, b u t
t h e s e a r e m o r e difficult t o u s e . I n a d d i t i o n ,
w a t e r i m m e r s i o n objectives h a v e been designed
t o o b s e r v e a q u a t i c o b j e c t s d i r e c t l y in t h e i r n a t u -
ral e n v i r o n m e n t . Objectives especially con-
s t r u c t e d for i m m e r s i o n in a n a q u e o u s s o l u t i o n of
g l y c e r i n o r c a n e s u g a r a r e u s e d for o b s e r v a t i o n s
u n d e r u l t r a v i o l e t l i g h t (Figure 1.14).
F o r a l o n g t i m e c e d a r oil ( n o t to b e c o n f u s e d
w i t h t h e e s s e n c e of t h e s a m e n a m e ) h a s b e e n
u s e d . N o w a d a y s , t h i s is b e i n g g r a d u a l l y r e -
130 -J nm p l a c e d b y s y n t h e t i c oils w h i c h a r e less v i s c o u s
Wavelength
a n d d o n o t solidify if left a c c i d e n t a l l y o n t h e
front l e n s . N e v e r t h e l e s s , it is r e c o m m e n d e d t h a t
t h e front l e n s b e w i p e d w i t h l e n s t i s s u e b e t w e e n
X observations b u t never with chamois leather.
R
- 2 O.N. S o l v e n t s w h i c h c o u l d l o o s e n lenses s h o u l d n o t b e
applied.
T h e r e f r a c t i v e i n d e x of t h e i m m e r s i o n oil is
Power
n o r m a l l y 1.515. E x c e s s i v e d e v i a t i o n from t h i s
of resolutton { / i m ) (
v a l u e c a u s e s a d e c r e a s e in t h e fineness of t h e
image. This sometimes happens when working
Figure 1.13. Nomogram for determining the power of
resolution of objectives. Above 1.0 , tenths of microns u n d e r e x t r e m e t e m p e r a t u r e s . O t h e r oils c a n b e
are used u s e d , w h e n i m m e r s i o n oil is n o t a v a i l a b l e . T h e s e
10 Instruments and techniques

F i g u r e 1.14. Light path in an objective.


Leftdry; rightimmersed, a, front of the
condenser; b, slide carrier; O, object; c,
object-covering slide; d, air or oil space;
e, front of objective

a r e c a s t o r oil ( = 1.478), e s s e n c e of cloves ( = T h e m i c r o s c o p e (Figures 1.15 a n d 1.16) m a y b e


1.533) o r g l y c e r i n s a t u r a t e d w i t h c a d m i u m r e g a r d e d a s a s o r t of ' o p t i c a l level a r m ' w h i c h ,
c h l o r i d e (n = 1.514). H o w e v e r , t h e s e s h o u l d b e c a u s e of its m a g n i f y i n g a c t i o n , e n l a r g e s n o t
o n l y b e u s e d as a last r e s o r t . o n l y t h e o b j e c t b u t also a n y i n s t a b i l i t y of t h e
stand. Obviously, one cannot obtain good im-
Antireflection treatment of optical surfaces a g e s if t h e s e a r e n o t m e c h a n i c a l l y s t a b l e . T h i s
s t a b i l i t y is g o v e r n e d b y t h e d e s i g n of t h e s t a n d ,
W h e n a p e n c i l of light i m p i n g e s o n a glass its m a n u f a c t u r e a n d t h e m a n n e r of its i n s t a l l a -
s u r f a c e , a l a r g e p a r t of it is refracted a n d p a s s e s t i o n w h i c h is f r e q u e n t l y c a r r i e d o u t w i t h insuffi-
t h r o u g h the glass. A smaller portion, a p p r o x - c i e n t c a r e . L a r g e m o d e r n r e s e a r c h s t a n d s often
i m a t e l y 6 - 8 % , is reflected, a n d lessens t h e h a v e a ' b r i d g i n g ' or m o d u l a r d e s i g n w h i c h
c o n t r a s t of t h e i m a g e o r e v e n p r o d u c e s a b r i g h t g u a r a n t e e s a d e g r e e of s t a b i l i t y n o t a t t a i n a b l e
s p o t a t its c e n t r e . S i n c e t h e o p t i c a l s y s t e m of with T - s h a p e d cross structures with a canti-
c u r r e n t m i c r o s c o p e s is m a d e u p of a b o u t 20 lever. T o test t h i s s t a b i l i t y in p r a c t i c e , t h e
a i r - g l a s s interfaces b e t w e e n t h e object a n d t h e following p o i n t s m u s t b e o b s e r v e d :
o b s e r v e r ' s eye, t h e q u a n t i t y of light lost is v e r y
c o n s i d e r a b l e . A t r a n s p a r e n t c o a t i n g of g i v e n
t h i c k n e s s , h a v i n g a refractive i n d e x less t h a n (1) S t a b i l i t y of m o v e m e n t w h e n t h e m i c r o -
t h a t of g l a s s , is d e p o s i t e d o n t h e g l a s s ; t h i s s c o p e c a r r i e s all its a c c e s s o r i e s , t h e 'drift'
r e d u c e s reflections to a c o n s i d e r a b l e e x t e n t , if m a y b e slow a n d it b e c o m e s a w k w a r d to u s e
t h e t h i c k n e s s of t h e c o a t i n g h a s a v a l u e of in c i n e m i c r o g r a p h y .
o n e - q u a r t e r of t h e w a v e l e n g t h of t h e i n c i d e n t (2) S t a b i l i t y of t h e o b s e r v a t i o n t u b e t h e t u b e
light. T h i s is c a u s e d b y a p h a s e shift b y / 2 in m u s t not bend when coming into contact
t h e reflection l e a d i n g to its e x t i n c t i o n , w h i c h with the observer's eyebrows.
o c c u r s u n d e r t h e s e c o n d i t i o n s . F r o m this it c a n (3) S t a b i l i t y of t h e s t a g e t h e s t a g e s h o u l d n o t
i m m e d i a t e l y b e d e d u c e d t h a t t o t a l e x t i n c t i o n is b e n d appreciably when the controls are
only possible at one particular wavelength. I n m a n i p u l a t e d o r w h e n t h e p r e p a r a t i o n is
g e n e r a l this lies a t a b o u t 0.55 , a n d t h e placed on the stage or moved by h a n d .
r e s i d u a l reflection a s s u m e s a p u r p l e t i n g e . M u l - (4) T h e r m a l s t a b i l i t y f o c u s i n g m u s t n o t b e
tiple c o a t i n g t r e a t m e n t m a k e s it p o s s i b l e to affected b y t e m p e r a t u r e v a r i a t i o n s d u r i n g
achieve practically complete extinction over the prolonged photography.
w h o l e s p e c t r a l r a n g e . T h i s is u s e d in p a r t i c u l a r
for i n c i d e n t i l l u m i n a t i o n a n d p o l a r i z a t i o n . T h i s s t a b i l i t y e n s u r e s a c e r t a i n d e g r e e of r e -
liability. A m o n g o t h e r factors, m e n t i o n s h o u l d
b e m a d e of t h e s t a b i l i t y of t h e l i g h t i n g s y s t e m ,
1.2 Basic instruments t h e a c c u r a c y of t h e e x p o s u r e m e t e r , a n d w h e t h e r
all t h e objectives u s e d a r e p a r f o c a l .
Mechanical features of the microscope
E a s e of u s e is o b t a i n e d b y a c o n s t r u c t i o n in
W h e n c o n s i d e r i n g t h e p o i n t of view of t h e u s e r which certain controls are a r r a n g e d coaxially
r a t h e r t h a n t h a t of t h e d e s i g n e r , t h r e e i m p o r t a n t a n d l o c a t e d a t t h e b a s e of t h e s t a n d so t h a t t h e
p r o p e r t i e s m u s t b e e x a m i n e d : s t a b i l i t y , reliabil- h a n d s a l w a y s rest o n t h e t a b l e . U n f o r t u n a t e l y ,
ity a n d c o n v e n i e n c e . t h e p r a c t i c e of fixing h e a d r e s t s to w a l l s , w h i c h is
Basic instruments 11

t h e o n l y r e m e d y a g a i n s t fatigue in t h e n e c k
during prolonged observation with the head
b e n t f o r w a r d , is n o t in g e n e r a l u s e . I t s h o u l d b e
n o t e d in p a s s i n g t h a t m o s t m i c r o s c o p e s h a v e
b i n o c u l a r t u b e s n o t p o s i t i o n e d sufficiently close
to the horizontal. T h e ideal position would be at
a n a n g l e of a b o u t 15 a n d n o t 45 o r e v e n 60 to
the horizontal.

Inverted m i c r o s c o p e s
T h e i n v e r t e d m i c r o s c o p e {Figure 1.17) w a s in-
v e n t e d b y L e C h a t e l i e r . I t is t h e m i c r o s c o p e
m o s t c o m m o n l y u s e d in m e t a l l u r g y . A l t h o u g h it
w a s o r i g i n a l l y d e v i s e d for m i c r o c h e m i s t r y , it h a s
p r o v e d v e r y useful in n u m e r o u s o t h e r fields;
a m o n g t h e s e a r e t h e e x a m i n a t i o n of c u l t u r e s in
flat-bottomed dishes (Petri dishes) or flasks,
m i c r o d i s s e c t i o n a n d m i c r o m a n i p u l a t i o n , ex-
a m i n a t i o n of c o r r o s i v e l i q u i d s , e x a m i n a t i o n of
p l a n t p a r a s i t e s , o b s e r v a t i o n of serological a g g l u -
tination, complement reactions, blocking reac-
Figure 1.15. Stiassnie research microscope with inclined t i o n s , m i c r o p r e c i p i t a t e s , m e a s u r e m e n t of m e l t -
binocular tube, mechanical stage with vertical and i n g p o i n t d e p r e s s i o n , e x a m i n a t i o n of d e n s e o b -
horizontal co-axial movement, and built-in illumination jects through an evacuated observation cham-
(SNM, Paris)
b e r , a n d e x a m i n a t i o n s a t low a n d h i g h t e m p e r a -
t u r e s , in sterilized c h a m b e r s , in a 'glove b o x ' for
r a d i o a c t i v e s u b s t a n c e s , a n d in d e n s e l i q u i d
media.
M o d e r n inverted microscopes allow the addi-
t i o n of a l a r g e n u m b e r of o p t i c a l a n d m e c h a n i c a l
accessories: p h a s e contrast, interference phase,
fluorescence, d a r k field, m i c r o d i s s e c t i o n , m i c r o -
m a n i p u l a t i o n . T o t a k e a c c o u n t of t h e t h i c k n e s s
of t h e o b j e c t s , t h e m i c r o s c o p e is fitted w i t h
long focal-length condensers a n d with long-
d i s t a n c e front lens o b j e c t i v e s , w i t h a c o r r e c t i o n
r i n g for t h i c k g l a s s .
W h e n observations are m a d e across the cul-
t u r e m e d i u m , t h e l a y e r of m e d i u m m u s t b e a s
t h i n a s p o s s i b l e . If t h e m e d i u m is a l i q u i d , a n
e l e c t r o n i c light is i n d i s p e n s a b l e for t a k i n g
photographs, because mechanical vibrations
would cause considerable disturbance.

Stereo-microscopes, the 'Microstereo-


projector' and microdissectors
S t e r e o - m i c r o s c o p e s {Figures 1.18-1.20) a r e often
i n c o r r e c t l y called b i n o c u l a r m a g n i f i e r s . T h e y
a r e d o u b l e m i c r o s c o p e s w i t h t w o objectives a n d
Figure 1.16. Portable Wild microscope with protective two eyepieces which have a permanently
metal cap (Wild-Heerbrugg) m o u n t e d d o u b l e p r i s m c a r r i a g e for a d j u s t i n g
12 Instruments and techniques

F i g u r e 1.19. B B T Strovar: a stereo-microscope with


magnification changer and with flexible fibre optic
illumination; this illumination gives a cold light and great
flexibility of orientation (BBT, Paris)

Figure 1.17. Inverted Wild microscope (Wild-Heerbrugg)

Figure 1.18. Simple Gruber LBB stereo-micoscope with F i g u r e 1.20. Wild stereo-microscope with magnification
light stand (Gruber) changer, showing the light path (Wild-Heerbrugg)
Basic instruments 13

images and producing a three-dimensional a d d e d a t least 18 h before it is to b e u s e d . T h e


effect. E v e n m o r e c a r e m u s t b e t a k e n in d e s i g n - f o r m a l d e h y d e v a p o u r e m i t t e d sterilizes t h e r e -
i n g t h e i r m e c h a n i c a l c o m p o n e n t s t h a n in t h e cesses of t h e a p p a r a t u s . T h e p l a s t i c b a g is
c a s e of s i n g l e - o b j e c t i v e m i c r o s c o p e s , a n d so t h e y r e m o v e d before u s e a n d t h e e y e p i e c e s a r e w i p e d
are more expensive. Objects can be illuminated w i t h a sterile p a d . P r o v i d e d h e w e a r s sterile
e i t h e r b y i n c i d e n t o r t r a n s m i t t e d light. V a r i o u s g l o v e s , t h e o p e r a t o r c a n t o u c h all c o m p o n e n t s
a c c e s s o r i e s a r e a v a i l a b l e for r e s e a r c h w i t h w i t h o u t risk of c o n t a m i n a t i n g t h e a p p a r a t u s .
stereo-microscopes: photographic equipment,
d u p l i c a t e o b s e r v a t i o n , d e v i c e s for t w o o p e r a t o r s
Principal light sources
working simultaneously, drawing tubes, micro-
dissectors, etc. DAYLIGHT
4
T h e M i c r o s t e r e o - p r o j e c t o r ' a l l o w s for c o n v e - T h i s l i g h t s o u r c e is h a r d l y e v e r u s e d b e c a u s e
nient stereoscopic examination by one or several t h e r e is t o o m u c h v a r i a t i o n in i n t e n s i t y a n d in
observers. I n this a p p a r a t u s , developed by V E B colour t e m p e r a t u r e . Nevertheless, research
C . Z e i s s of J e n a , t h e t w o i m a g e s c o m i n g from w o r k e r s c o n s i d e r d a y l i g h t to b e i r r e p l a c e a b l e for
t h e t w o o b j e c t i v e s a r e p r o j e c t e d o n to a p i e c e of r e v e a l i n g a n a m y l o i d r e a c t i o n to i o d i n e .
g r o u n d glass c o u p l e d w i t h a F r e s n e l l e n s w h i c h
receives t h e e n l a r g e d a n d diffuse i m a g e from t h e GAS LAMPS
t w o exit p u p i l s of t h e o p t i c a l s y s t e m . T h u s t h e I n a r e a s w i t h o u t a n electricity s u p p l y , a g a s
p o s i t i o n of t h e o b s e r v e r ' s eyes is n o t v e r y c r i t i c a l l a m p with an Auer mantle can be used. T h e
a n d o b s e r v a t i o n b e c o m e s c o m f o r t a b l e . T h e fact b r i g h t n e s s is sufficient for e x a m i n a t i o n a g a i n s t a
that the image can be quickly viewed by m a n y b r i g h t b a c k g r o u n d , b u t t h e g r e e n i s h t i n g e of t h e
observers means that demonstration and control light alters the colours.
can be carried out.
M i c r o d i s s e c t o r s a r e simplified m i c r o m a n i p u - I N C A N D E S C E N T BULBS
l a t o r s w h i c h a l l o w for s i m p l e o p e r a t i o n s a t l o w T h e h e a t i n g of a t u n g s t e n f i l a m e n t b y t h e J o u l e
m a g n i f i c a t i o n (cf. p . 7 8 ) . effect r e m a i n s t h e m o s t c o m m o n light s o u r c e .
F o r t h e l i g h t i n g to b e effective, t h e f i l a m e n t m u s t
STERILIZATION b e a s c o m p a c t a s p o s s i b l e . F o r t h i s , a low
S t e r e o - m i c r o s c o p e s a r e f r e q u e n t l y u s e d in s u r - v o l t a g e o f 6 - 1 2 V is u s e d w i t h f l a t t e n e d coils o r
gical o p e r a t i o n s , for e x a m p l e in o p h t h a l m o l o g y . a grid. T h e l a m p m a y be furnished with an
T h e a p p a r a t u s m u s t first b e sterilized. H e a t adjustable mirror, which must not turn the
c a n n o t b e u s e d , b e c a u s e t h i s w o u l d loosen t h e i m a g e of t h e f i l a m e n t b a c k o n itself, b u t to o n e
lenses, nor can sterilization by ultraviolet light, s i d e so a s to d o u b l e t h e useful surface. Q u a r t z -
b e c a u s e it d o e s n o t p e n e t r a t e i n t o t h e r e c e s s e s of iodine or quartz-halogen bulbs are equipped
the mechanism and into the tubes. T h e best w i t h a s m a l l q u a r t z b u l b b e c a u s e t h e p r e s e n c e of
m e t h o d was p u t forward by F. B o c k m h l a n d R. i o d i n e a n d b r o m i n e w i t h k r y p t o n in t h e v a p o u r
K o s s e k . T h e w h o l e i n s t r u m e n t is p l a c e d in a w i t h i n t h e b u l b p r e v e n t s t h e e v a p o r a t i o n of t h e
p l a s t i c b a g a n d 5 g of p a r a f o r m a l d e h y d e a r e t u n g s t e n from t h e f i l a m e n t , a n d t h u s p e r m i t s it

BS
100

80
QI
8V
60

40
6V

20 5V
F i g u r e 1.21. Relative spectral brightness of
tungsten filament bulbs. 6 V 30 W, supplied
0|
by 5, 6 and 8 V and QI: quartz-iodine 12 V
400 500 600 700 800 900 1000 1100 1200
300 lOOWat 12 V

14 Instruments and techniques

to function at m u c h higher t e m p e r a t u r e s . T h e ARC LAMPS IN A G A S E O U S A T M O S P H E R E


r e s u l t i n g c o l o u r t e m p e r a t u r e is also h i g h e r W h e r e a s t h e r m a l e m i s s i o n from a h e a t e d b o d y is
{Figure 1.21). u s e d in t h e t w o p r e c e d i n g e x a m p l e s , this t y p e of
a r c l a m p utilizes t h e e m i s s i o n of g a s e x c i t e d b y
ARC LAMPS BETWEEN T U N G S T E N SPHERES
t h e p a s s a g e of c u r r e n t in t h e a r c . T h i s r e s u l t s in
T h e b r i g h t n e s s of t h e a r c is n o t u s e d as t h e light
a s p e c t r u m of r a y s w h i c h a r e m o r e o r less
s o u r c e , b u t t h e b r i g h t n e s s of o n e of t h e t w o
superimposed on a continuous spectrum, de-
s p h e r e s b r o u g h t to i n c a n d e s c e n c e . T h e i r p o w e r
p e n d i n g o n t h e e x c i t a t i o n c u r r e n t a n d its r e s o -
is fixed a n d c a n n o t b e a d j u s t e d .
n a n c e w i t h t h e g a s m o l e c u l e s . T h u s , perfectly
C O N C E N T R A T E D ARC Z I R C O N I U M LAMPS m o n o c h r o m a t i c s o u r c e s , s u i t a b l e for m i c r o i n t e r -
A s m a l l c r u c i b l e filled w i t h z i r c o n i u m is h e a t e d f e r o m e t e r s o r v e r y p o w e r f u l s o u r c e s e m i t t i n g in
b y a n a r c s p a r k i n g b e t w e e n its surface a n d a t h e visible, u l t r a v i o l e t o r i n f r a r e d r e g i o n s , c a n b e
r i n g - s h a p e d e l e c t r o d e . T h e m o l t e n surface of t h e obtained. Sodium a n d mercury vapour are the
z i r c o n i u m r e t a i n e d b y a t r a n s p a r e n t surface film t w o g a s e s m o s t f r e q u e n t l y u s e d , followed b y
of z i r c o n i u m o x i d e a c t s as t h e s o u r c e . T h e x e n o n , a n d a r e e m p l o y e d w h e n it is d e s i r e d to
b r i g h t n e s s o b t a i n e d is v e r y h i g h a n d t h e s o u r c e emphasize the continuous background. T h e
in t h e form of a v e r y s m a l l c i r c u l a r a r e a is v e r y w a l l s of t h e b u l b a r e m a d e of q u a r t z for w o r k
homogeneous. o u t s i d e t h e visible r a n g e of t h e s p e c t r u m . T h e

BM aBP BP

50
300
20 200

200 30


u J
10 20 -100
100

Vi w
200 300 400 500 600 700 800 900 1000 200 300 400 500 600 700 800 900 1000
[nm] [nm]

Figure 1.22. Spectral distribution of the energy from a F i g u r e 1.24. Spectral energy distribution of the xenon lamp,
mercury vapour lamp, Leitz HG 100W (Leitz-Wetzlar) Leitz 150W (Leitz-Wetzlar)

F i g u r e 1.23. Spectral
emission curve for the
mercury and metal iodide
Philips CSI lamp (Philips)
Basic instruments 15

IllllllllllllttllltWJ
In i n f i n i , !

I Y/
\ 1
a
11
0 >PAM

HBO 2 0 0 W/2
HBO 5 0 W/AC "S HBO 2 0 0 W/4
HBO 2 0 0 W
HBO 5 0 W/3 JHIIIIIIIIIIIIIIH
HBO 5 0 0 W/2
_ 9
1 4 5

6 7 8 9 10 11

( 6 4 2 6 0 Lnge max 4 0 m m )

Figure 1.25. Different types of Osram bulb. Toparc burners in a gaseous atmosphere; bottom leftlow voltage
filament bulbs; bottom rightquartz-halogen bulb (Osram)

internal electrodes are m a d e of t u n g s t e n or


c a d m i u m (Figures 1.22-1.26).

Electronic flashlights for photomicrography


T h e m i c r o s c o p e u s e r s w i t c h i n g to p h o t o m i c -
r o g r a p h y quickly encounters two limitations:
t h e d e c r e a s e in t h e a m o u n t of l i g h t r e c e i v e d o n
t h e s e n s i t i v e s u r f a c e , w h i c h d e c r e a s e s in i n v e r s e
p r o p o r t i o n to t h e s q u a r e of t h e m a g n i f i c a t i o n ,
a n d t h e i n s t a b i l i t y w h i c h is a m p l i f i e d b y t h e
' o p t i c a l lever a r m ' of t h e m a g n i f i c a t i o n . T h i s
i n s t a b i l i t y m a y b e i n t r i n s i c to t h e o b j e c t o r m a y
b e d u e to s o m e i n c i d e n t a l v i b r a t i o n s . T o over-
c o m e these two limitations, microscope desig-
n e r s h a v e l o o k e d for light s o u r c e s of e v e r in-
c r e a s i n g b r i g h t n e s s . C o n t i n u o u s i l l u m i n a t i o n of
the object with these new sources leads, howev-
Figure 1.26. Illumination stand fitted with high power
lamps for research microscopes (Wild-Heerbrugg) e r , to p r o b l e m s s u c h a s a l e t h a l a c t i o n o n living
16 Instruments and techniques

o b j e c t s o r f a d i n g of c o l o u r e d o b j e c t s . I n p r i n c i - M a n y people hold t h e m by the a r m which


p l e , e l e c t r o n i c flashlights h e l p to r e m e d y t h e s e s u p p o r t s t h e t u b e ; t h i s is a d e p l o r a b l e p r a c t i c e
d r a w b a c k s , b e c a u s e t h e e x p o s u r e t i m e of t h e a n d s o o n e r o r l a t e r l e a d s to a d e t e r i o r a t i o n of t h e
o b j e c t a t s u c h a h i g h l u m i n a n c e is o n l y a few m i c r o m e t e r screw. Indeed, w h e n grasping the
t h o u s a n d t h s of a s e c o n d . T h e n e e d for c o m b i n - m i c r o s c o p e in this w a y , t h e w h o l e w e i g h t of t h e
i n g a c o n t i n u o u s i l l u m i n a t i o n for o b s e r v a t i o n a p p a r a t u s rests o n t h e s c r e w a n d t h e t h r e a d
w i t h v e r y s h o r t i l l u m i n a t i o n for t a k i n g p h o t o - gradually becomes worn away. Obviously, a
g r a p h s led d e s i g n e r s to b u i l d c o m b i n e d d e v i c e s microscope m u s t never be held by the stage or
in w h i c h t h e flash is e i t h e r i n t e r p o s e d o r in the tube.
p a r a l l e l w i t h t h e n o r m a l i l l u m i n a t i o n of t h e
microscope. Adjustments, observations and exp-
osure m e a s u r e m e n t s are carried out with con- Positioning of the microscope
tinuous illumination, supplied at constant a n d T h e w o r k i n g life a n d to s o m e e x t e n t t h e d e g r e e
known voltage. For most purposes, flashlights of c o m f o r t w h e n u s i n g a m i c r o s c o p e d e p e n d s o n
delivering 1 0 0 - 4 0 0 J are recommended. T h e its efficient i n s t a l l a t i o n . L a b o r a t o r y a n d r e -
c o l o u r t e m p e r a t u r e of t h e lights is 5 6 0 0 K ; as s e a r c h m i c r o s c o p e s a r e g e n e r a l l y d e l i v e r e d in
d i s c u s s e d e a r l i e r o n , it c a n b e modified b y t h e s m a l l b o x e s . T h e s e b o x e s s h o u l d b e k e p t careful-
o p t i c a l s y s t e m of t h e m i c r o s c o p e , b e c a u s e glass ly a n d n o t d i s c a r d e d . T h e i n s t r u m e n t c a n b e p u t
is n o t a l w a y s c o l o u r l e s s . I t m u s t b e p o s s i b l e to b a c k i n t o its b o x for t r a n s p o r t or d u r i n g p r o -
c o n n e c t u p a n i n d u c t i o n coil in series w i t h t h e l o n g e d p e r i o d s of n o n - u s e . I n fact, d u s t is a g r e a t
t u b e i n p u t so t h a t t h e d u r a t i o n of t h e flash d o e s e n e m y of o p t i c a l i n s t r u m e n t s , a n d bells o r cov-
n o t fall b e l o w 0.002 s to p r e v e n t t h e S c h w a r z - ers which m u s t be placed over the a p p a r a t u s
s c h i l d effect from c o m i n g i n t o p l a y . T h i s effect each evening are not dust-proof.
t e n d s to r e d u c e t h e efficiency of t h e sensitive I n c h o o s i n g t h e p o s i t i o n of t h e m i c r o s c o p e ,
l a y e r for s h o r t e x p o s u r e t i m e s b y r e d u c i n g sensi- t h e following factors m u s t b e t a k e n i n t o c o n s i d -
tivity a n d c o n t r a s t . e r a t i o n : t h e least p o s s i b l e floor v i b r a t i o n s ; t h e
Some automatic chambers have had incorpo- a m b i e n t light s h o u l d n o t b e excessively b r i g h t ;
r a t e d a s y s t e m w h i c h a l l o w s t h e i n p u t to t h e t h e i n s t r u m e n t s h o u l d p r e f e r a b l y p o i n t to t h e
t u b e to b e c u t off a u t o m a t i c a l l y a s s o o n as t h e n o r t h ; its s u p p o r t s h o u l d b e fixed to a t a b l e o r to
o p t i m u m a m o u n t of light h a s r e a c h e d t h e sensi- t h e w a l l ; t h e i n s t r u m e n t s h o u l d face a w a l l
tive l a y e r . If this e q u i p m e n t is n o t a v a i l a b l e , r a t h e r t h a n a w i n d o w , u n l e s s d a y l i g h t is g o i n g to
trials m u s t b e c a r r i e d o u t w i t h a test p r e p a r a - b e u s e d d u r i n g o b s e r v a t i o n s ; t h e h e i g h t of t h e
t i o n , w i t h different s e t t i n g s r e c o r d i n g t h e r e a d - b e n c h a n d of t h e stool s h o u l d c o r r e s p o n d to t h e
ings given by the m e a s u r i n g i n s t r u m e n t s , a n d h e i g h t of t h e o p e r a t o r , t h e h e i g h t of t h e m i c r o -
u s i n g c o n t i n u o u s i l l u m i n a t i o n for t h e o b s e r v a - s c o p e a n d t h e a n g l e of i n c l i n a t i o n of t h e o b s e r v a -
t i o n . A r a n g e of n e u t r a l filters is u s e d to r e d u c e tion or sighting tube. T h e l a m p lighting the
t h e i n t e n s i t y a t low m a g n i f i c a t i o n s . w o r k i n g surface should be e q u i p p e d with a n
a p p l i a n c e for v a r y i n g t h e i n t e n s i t y , so t h a t t h e r e
will b e u n i f o r m i l l u m i n a t i o n o n t h e field
Use of the microscope
o b s e r v e d a n d o n t h e p a p e r u s e d for d r a w i n g o r
T h e c o n c i s e h i n t s given h e r e o n t h e u s e of m a k i n g n o t e s . T h i s is especially i m p o r t a n t if
microscopes are very necessary. T h e most drawings are m a d e with a drawing a p p a r a t u s .
e l e m e n t a r y r u l e s for h a n d l i n g m i c r o s c o p e s a r e
often i g n o r e d , e v e n b y q u i t e e x p e r i e n c e d r e -
Installation of the microscope
search workers w h o , t h r o u g h careless h a n d l i n g ,
s h o r t e n t h e w o r k i n g life of t h e i r i n s t r u m e n t s o r T h e m i c r o s c o p e , c a r r i e d c o r r e c t l y , is t a k e n o u t
d e p r i v e t h e m s e l v e s of s o m e of t h e i r facilities. of t h e c u p b o a r d a n d p l a c e d o n a t a b l e . I t is
a d v i s a b l e to p l a c e it o n a s h e e t of felt o r r u b b e r
Method of h o l d i n g and transporting o r a b o o k of filter p a p e r to e n s u r e its s t a b i l i t y
microscopes a n d to p r e v e n t it from s l i p p i n g . T h e n t h e o b j e c -
T h e c o r r e c t m e t h o d of c a r r y i n g m i c r o s c o p e s is tives a r e s c r e w e d o n to t h e r e v o l v i n g n o s e p i e c e ,
i m p o r t a n t in p r e v e n t i n g d a m a g e to t h e m i c r o - t a k i n g c a r e to p l a c e t h e m in o r d e r of t h e i r
meter screw. Microscopes with horizontal mic- p o w e r . I t is a g o o d i d e a to s c r e w in t h e o b j e c -
rometric m o v e m e n t m u s t be held by the base. tives in s u c c e s s i o n , so t h a t t h e y a r e a r r a n g e d
Basic instruments 17

a c c o r d i n g to o b s e r v a t i o n r e q u i r e m e n t s . W h e n a x i s . I f t h e p r e p a r a t i o n is o n l y c o m p o s e d of a
t h i s is d o n e , t h e first o b j e c t i v e to b e b r o u g h t i n t o few, v e r y w i d e l y s e p a r a t e d o b j e c t s , focusing c a n
t h e o p t i c a l axis will b e low p o w e r . T h e e y e p i e c e either be m a d e on a n air b u b b l e or on the edge
is r e m o v e d a n d t h e l i g h t i n g is a d j u s t e d a s d e - of t h e c o v e r - s l i p . T h u s t h e w o r k i n g d i s t a n c e of
scribed below, by directing the mirror towards t h e low p o w e r o b j e c t i v e c a n b e f o u n d a n d , b y
t h e light s o u r c e a n d f o c u s i n g t h e c o n d e n s e r . T h e s y s t e m a t i c a l l y d i s p l a c i n g t h e p r e p a r a t i o n , o n e of
l i g h t i n g a d j u s t m e n t is c o m p l e t e d b y p l a c i n g a t h e o b j e c t s o n t h e slide c a n s o o n b e b r o u g h t i n t o
l o w p o w e r e y e p i e c e in t h e t u b e . t h e c e n t r e of t h e field of vision. B y c o m b i n i n g
F o r m i c r o s c o p e o b s e r v a t i o n it is p r e f e r a b l e to these m o v e m e n t s , the low p o w e r objective can
w o r k far a w a y from t h e w i n d o w a n d o n a d a r k q u i c k l y b e focused.
t a b l e to e l i m i n a t e all e x t r a n e o u s light w h i c h B y m e a n s of t h e r e v o l v i n g n o s e p i e c e , it is
d a z z l e s a n d interferes w i t h g o o d i m a g e defini- e x t r e m e l y s i m p l e t o p r o g r e s s to a h i g h e r p o w -
t i o n . T h e s e p r e c a u t i o n s r e s u l t in less f a t i g u e . e r e d o b j e c t i v e . W h e n t h e m i c r o s c o p e is well
a d j u s t e d , all t h a t n e e d s to b e d o n e is to t u r n t h e
r e v o l v i n g n o s e p i e c e a n d slightly modify t h e
F o c u s i n g the m i c r o s c o p e
focus b y m e a n s of t h e fine a d j u s t m e n t .
F i r s t , a stool is c h o s e n of t h e r i g h t h e i g h t to B y following t h e s e i n s t r u c t i o n s , t h e b e g i n n e r
e n a b l e t h e o b s e r v e r to look i n t o t h e m i c r o s c o p e c a n b e c e r t a i n of d a m a g i n g n e i t h e r t h e p r e p a r a -
w i t h o u t u n d u e f a t i g u e . A p r e p a r a t i o n is t h e n tions n o r the objectives, a n d this p r o c e d u r e
placed on the microscope stage. A table giving s h o u l d b e followed b y all m i c r o s c o p i s t s . W i t h
t h e w o r k i n g d i s t a n c e s a n d t h e m a g n i f i c a t i o n s of practice, these movements can be performed
t h e objectives s h o u l d b e d i s p l a y e d . T h u s , before v e r y q u i c k l y : o n l y b y s t r i c t a d h e r e n c e to t h e s e
placing the preparation on the microscope stage, p r o c e d u r e s from t h e b e g i n n i n g c a n o n e l e a r n t h e
c a r e is t a k e n to r a i s e t h e m i c r o s c o p e t u b e b y t h e s y s t e m a t i c e x a m i n a t i o n of a p r e p a r a t i o n . F r e -
r a t c h e t to a h e i g h t g r e a t e r t h a n t h e w o r k i n g q u e n t l y , o n e h a s s e e n a n e x a m i n a t i o n of a
d i s t a n c e of t h e low p o w e r o b j e c t i v e . W h e n this is s e c t i o n s t a r t e d u s e d a m a g n i f i c a t i o n of 3 0 0 o r
d o n e , a s e c t i o n of t h e p r e p a r a t i o n clearly visible 4 0 0 d i a m e t e r s , o r e v e n w i t h a n oil i m m e r s i o n
t o t h e n a k e d eye is p l a c e d in t h e c e n t r e of t h e o b j e c t i v e . T h i s lack of a s y s t e m a t i c a p p r o a c h
o p e n i n g in t h e s t a g e . T h e o b s e r v e r t h e n looks h a s c a u s e d t h e g r o s s e r r o r s t h a t t e n d to u n d e r -
t h r o u g h t h e e y e p i e c e a n d t h e t u b e is v e r y slowly m i n e t h e c o n f i d e n c e in t h e i n f o r m a t i o n p r o v i d e d
lowered by the coarse adjustment. As soon as by the microscope. O n the other hand, by
t h e i m a g e a p p e a r s , t h e r a t c h e t m o v e m e n t is b e g i n n i n g t h e e x a m i n a t i o n of a p r e p a r a t i o n w i t h
s t o p p e d a n d focusing is c o m p l e t e d b y o p e r a t i n g a l o w p o w e r m a g n i f i c a t i o n , focusing is m a d e
t h e fine a d j u s t m e n t . m u c h e a s i e r , a n o v e r a l l v i e w of t h e l a y o u t c a n b e
W h e n t h e fine a d j u s t m e n t is n o t of t h e e n d l e s s m a d e a n d t h e i n t e r e s t i n g p a r t s of t h e p r e p a r a -
v a r i e t y , c a r e m u s t b e t a k e n a l w a y s to k e e p it t i o n c a n b e l o c a t e d w i t h o u t difficulty. T h e n ,
n e a r t h e m i d d l e of its c o u r s e a n d to a l l o w it o n l y e x a m i n a t i o n w i t h m o r e p o w e r f u l objectives c a n
to u n d e r g o s m a l l d i s p l a c e m e n t s . If it is a l l o w e d be undertaken.
to r e a c h t h e e n d of its c o u r s e , focusing m a y
b e c o m e i m p o s s i b l e or, if d o n e p e r s i s t e n t l y , t h e
F o c u s i n g with i m m e r s i o n objectives
thread may be stripped away.
If t h e i m a g e d o e s n o t a p p e a r , g r e a t c a r e m u s t T h e u s e of i m m e r s i o n o b j e c t i v e s r e q u i r e s s o m e
b e t a k e n n o t to d r i v e t h e o b j e c t i v e t h r o u g h t h e e x p l a n a t i o n b e c a u s e t h e y c a n n o t b e focused
p r e p a r a t i o n b y excessive d o w n w a r d m o v e m e n t simply by turning the revolving nosepiece. First
of t h e t u b e . T h i s m a y s h a t t e r t h e p r e p a r a t i o n of all o n e m a k e s s u r e t h a t t h e r e is s o m e t h i n g in
a n d c a n s e r i o u s l y d a m a g e t h e front l e n s . T h e r e - t h e field of vision u s i n g a l o w p o w e r o b j e c t i v e ;
fore, if n o i m a g e a p p e a r s , o n e s h o u l d m a k e t h i s is a n e s s e n t i a l p r e c a u t i o n b e c a u s e it is v e r y
absolutely certain, by examining the microscope difficult to s e a r c h for t h e object w i t h i m m e r s i o n
from o n e side, t h a t t h e limit of t h e w o r k i n g o b j e c t i v e s o n a c c o u n t of t h e low w o r k i n g d i s -
d i s t a n c e h a s n o t b e e n e x c e e d e d . If t h i s is t h e t a n c e a n d t h e n a r r o w n e s s of t h e field of vision.
case, the tube m u s t be raised u p a n d the d o w n - F u r t h e r m o r e , t h e low p o w e r o b j e c t i v e is e s s e n -
w a r d m o v e m e n t s t a r t e d a g a i n . If t h e r e is still n o tial for p r e l i m i n a r y r e g u l a t i o n of t h e i l l u m i n a -
i m a g e , t h e p r e p a r a t i o n m u s t b e d i s p l a c e d slight- tion.
ly to b r i n g t h e object d i r e c t l y i n t o t h e o p t i c a l T h u s , o n c e t h e i l l u m i n a t i o n a n d t h e object to
18 Instruments and techniques

b e e x a m i n e d a r e w e l l - c e n t r e d , t h e t u b e is r a i s e d , p r o b l e m o c c u r s especially w i t h h i g h p o w e r
t h e i m m e r s i o n o b j e c t i v e is b r o u g h t i n t o line w i t h o b j e c t i v e s a n d c a n b e d u e to o n e of t h r e e
t h e o p t i c a l axis a n d a d r o p of i m m e r s i o n oil is p r i n c i p a l c a u s e s (see b e l o w for o t h e r c a u s e s of
t h e n p l a c e d in t h e c e n t r e of t h e p r e p a r a t i o n . A s failure).
far a s p o s s i b l e , t h i s oil s h o u l d b e t h e o n e s u p -
p l i e d b y t h e m a n u f a c t u r e r s of t h e o b j e c t i v e , t h e T H E R E V O L V I N G NOSEPIECE IS OFF-CENTRE
refractive i n d e x of w h i c h h a s b e e n c a l c u l a t e d for T h i s o c c u r s w h e n t h e object c a n n o t b e f o u n d
t h e c o r r e c t i o n of t h e l e n s e s . a g a i n a t h i g h p o w e r m a g n i f i c a t i o n after h a v i n g
W h e n this h a s b e e n d o n e , t h e t u b e is l o w e r e d b e e n p o s i t i o n e d in t h e c e n t r e of t h e field of vision
for c o a r s e focusing w h i l e l o o k i n g from t h e side a t u n d e r low p o w e r m a g n i f i c a t i o n . T h i s p r o b l e m
t h e m i c r o s c o p e in o r d e r to b r i n g t h e front lens a r i s e s in p a r t i c u l a r w h e n e x a m i n i n g a p r e p a r a -
i n t o c o n t a c t w i t h t h e oil d r o p l e t . T h e l a t t e r tion containing very small organisms which are
s h o u l d h a v e r e t a i n e d its c o n v e x i t y ; therefore, t h e w i d e l y s p a c e d . T o p r e v e n t a c e n t r i n g defect,
i n v e s t i g a t o r m u s t n o t w a i t for it to s p r e a d o u t w h i c h m a y b e i n h e r e n t e v e n in t h e b e s t -
o v e r t h e p r e p a r a t i o n , u n l e s s h e a p p l i e s a little c o n s t r u c t e d r e v o l v i n g n o s e p i e c e s , it is n e c e s s a r y
m o r e oil. A s s o o n as c o n t a c t h a s b e e n e s t a b - t o fix t h e o b j e c t b y successive c e n t r i n g , first w i t h
l i s h e d a n d a c h a n g e in t h e s h a p e of t h e d r o p is a v e r y low p o w e r o b j e c t i v e , t h e n w i t h a m e d i u m
p e r c e i v e d , t h e t u b e is r a i s e d slightly w i t h o u t , p o w e r o n e . T h i s gives a b e t t e r c h a n c e of finding
h o w e v e r , b r e a k i n g c o n t a c t . O b s e r v a t i o n is t h e n it a g a i n w i t h a p o w e r f u l o b j e c t i v e . By c e n t r i n g
m a d e t h r o u g h the eyepiece, lowering the tube t h e o b j e c t i v e s successively, t h e e x t e n t to w h i c h
v e r y slowly w i t h t h e c o a r s e focus; a s s o o n a s t h e t h e p r e p a r a t i o n m u s t b e d i s p l a c e d to a c h i e v e
i m a g e a p p e a r s , e v e n a diffuse o n e , focusing is final c e n t r i n g c a n also b e d e t e r m i n e d , p r o g r e s -
c o m p l e t e d w i t h t h e fine a d j u s t m e n t . s i n g from a low p o w e r to a h i g h e r p o w e r o b j e c -
W h e n t h e o b s e r v a t i o n is finished, it is e s s e n - tive.
tial to r a i s e t h e t u b e w i t h t h e c o a r s e a d j u s t m e n t
T H E PREPARATION IS U P S I D E D O W N
in o r d e r to r e m o v e t h e front lens from t h e
T h i s minor accident occurs quite commonly
preparation. Only then can the preparation be
e v e n w i t h e x p e r i e n c e d m i c r o s c o p i s t s ; it is p a r t i -
w i t h d r a w n w i t h o u t fear of d a m a g i n g t h e o b j e c -
c u l a r l y c o m m o n w i t h b l o o d slides a n d d r y
tive b y r u b b i n g it a g a i n s t t h e c o v e r - s l i p . T h i s
C a n a d a b a l s a m p r e p a r a t i o n s . A t low p o w e r
p r e c a u t i o n is also n e c e s s a r y w i t h d r y h i g h p o w e r
m a g n i f i c a t i o n t h e e r r o r is n o t p e r c e i v e d a n d o n e
objectives.
is s u r p r i s e d a t n o t b e i n g a b l e to focus w i t h t h e
C L E A N I N G OF I M M E R S I O N OBJECTIVES oil i m m e r s i o n o b j e c t i v e o r e v e n w i t h a h i g h
A s s o o n a s t h e o b s e r v a t i o n is finished, oil a d h e r - p o w e r dry objective. I n d e e d , starting with a
i n g to t h e front lens m u s t b e r e m o v e d . I t is v e r y m a g n i f i c a t i o n of a p p r o x i m a t e l y 300, t h e w o r k -
b a d p r a c t i c e to l e a v e this oil to d r y u p o v e r n i g h t , i n g d i s t a n c e is t o o s h o r t for focusing o n a n o b j e c t
b e c a u s e t h e r e is t h e risk of s c r a t c h i n g t h e lens p l a c e d u n d e r a slide. I n this c a s e it is n e v e r
w h e n w i p i n g it, o r a p o r t i o n of t h e s o l v e n t u s e d a d v i s a b l e to p e r s i s t . I n s t e a d t h e t u b e m u s t b e
m a y p e n e t r a t e into the objective. Oil m u s t r a i s e d i m m e d i a t e l y a n d it s h o u l d b e c h e c k e d
t h e r e f o r e b e w i p e d off g e n t l y w i t h a p i e c e of lens t h a t t h e p r e p a r a t i o n is n o t u p s i d e d o w n . T o
p a p e r . N e v e r d i p t h e e n d of t h e o b j e c t i v e i n t o avoid this mistake, one should acquire the h a b i t
t h e l i q u i d s ; t h i s is a v e r y d a n g e r o u s p r a c t i c e of c h e c k i n g t h e p r o j e c t i o n of t h e c o v e r - s l i p w i t h
w h i c h c a n c a u s e t h e lens to b e c o m e u n g l u e d . a finger before p l a c i n g t h e p r e p a r a t i o n o n t h e
Some people have mistakenly suggested remov- m i c r o s c o p e s t a g e . F o r s m e a r s , t h e reflection of
i n g t h e oil w i t h s o l v e n t s . T h e s e l i q u i d s d i s s o l v e t h e p r e p a r a t i o n is e x a m i n e d to m a k e c e r t a i n
t h e c e m e n t u s e d to b o n d t h e front lens a n d t h u s t h a t t h e d u l l side is o n t o p .
d a m a g e the objective irreparably. Therefore,
ethanol, benzene, xylene or toluene should never T H E COVER-SLIP IS T O O T H I C K OR T H E R E ARE
b e u s e d to c l e a n i m m e r s i o n o b j e c t i v e s . T W O COVER-SLIPS S U P E R I M P O S E D
T h i s gives rise to t h e s a m e p h e n o m e n o n a s for
a n u p s i d e - d o w n p r e p a r a t i o n . T h e front lens
Causes of error in focusing knocks against the cover-slip a n d the i m a g e
either does not a p p e a r or remains u n s h a r p .
I n s p i t e of all p r e c a u t i o n s , it s o m e t i m e s h a p p e n s T h i s e r r o r o c c u r s m u c h less f r e q u e n t l y t h a n
t h a t n o i m a g e is o b t a i n e d w h e n focusing. T h i s formerly, because m o d e r n objectives, even high
Basic instruments 19

p o w e r o n e s , h a v e sufficient w o r k i n g d i s t a n c e s to e a s y to g e t i n t o t h e h a b i t of o b s e r v i n g w i t h b o t h
o v e r c o m e this inconvenience; also, m o d e r n eyes o p e n . After a s h o r t t i m e , o n l y t h e i m a g e s
cover-slips available commercially are m o r e uni- s u p p l i e d b y t h e eye l o o k i n g d o w n i n t o t h e
f o r m in t h i c k n e s s t h a n p r e v i o u s l y . N e v e r t h e l e s s , m i c r o s c o p e a r e p e r c e i v e d . H o w e v e r , it is e s s e n -
w h e n t h e p r e p a r a t i o n is v e r y t h i c k , it m a y tial t h a t t h e t a b l e o n w h i c h t h e i n s t r u m e n t is
h a p p e n t h a t o n e c a n n o t focus o n t h e d e e p e r r e s t i n g is n o t i l l u m i n a t e d m o r e b r i g h t l y t h a n t h e
l a y e r s , e v e n w i t h m o d e r n o b j e c t i v e s . I t is e s s e n - o p t i c a l field in t h e m i c r o s c o p e . T h e r e f o r e , if t h e
tial n o t to p e r s i s t in l o w e r i n g t h e t u b e , b e c a u s e t a b l e is w h i t e ( e n a m e l l e d ) o r b r i g h t l y c o l o u r e d ,
t h e c o v e r - s l i p will p r o b a b l y b e c r a c k e d , t h e it m u s t b e c o v e r e d w i t h d a r k o r b l a c k p a p e r . I n
o b j e c t c r u s h e d a n d p e r h a p s t h e front lens i r r e - t h i s w a y , t h e eye n o t b e i n g u s e d for o b s e r v a t i o n
parably damaged. It may be added that with the r e c e i v e s o n l y a s m a l l a m o u n t of light a n d forms
m o d e r n fine a d j u s t m e n t s , b r e a k a g e of t h e c o v e r - i m a g e s w h i c h a r e t o o d i m to interfere w i t h t h e
slip is m u c h less likely to o c c u r b e c a u s e , g e n e r a l - p e r c e p t i o n of t h e o t h e r eye.
ly, t h e s c r e w s t o p s m o v i n g t h e t u b e a s s o o n a s
the objective comes into contact with the cover- EYESHIELDS
slip. W h e n prolonged observations at high magni-
fications h a v e to b e c a r r i e d o u t , it is a n a d v a n -
t a g e to p r o t e c t b o t h eyes from e x t r a n e o u s light,
T h e fine adjustment and microscopic i.e. l i g h t w h i c h d o e s n o t o r i g i n a t e from t h e
observation microscope. Eyeshields a n d eyepiece shades
h a v e b e e n d e s i g n e d for this p u r p o s e . T h e y a r e
During observation with the microscope, one i n t e n d e d b o t h to p r e v e n t i m a g e s from f o r m i n g in
eye is p o s i t i o n e d o v e r t h e e y e p i e c e , o n e h a n d is t h e eye w h i c h is n o t u s e d in o b s e r v a t i o n a n d to
continually adjusting the micrometer screw a n d p r o t e c t t h e eye w h i c h is o b s e r v i n g from r a y s of
t h e o t h e r is slowly m o v i n g t h e p r e p a r a t i o n b y l i g h t reflected b y t h e m i c r o s c o p e m o u n t i n g , o r
u s i n g t h e c o n t r o l s of t h e s t a g e . d i r e c t l y o r i g i n a t i n g from t h e light s o u r c e .
T h e s i m p l e s t s h i e l d is m a d e w i t h a t h i n s h e e t
W H I C H EYE S H O U L D BE U S E D W H E N L O O K I N G
of b l a c k e n e d c a r d b o a r d w h i c h is c u t i n t o t h e
T H R O U G H A M O N O C U L A R MICROSCOPE?
s h a p e of t h e o r b i t of t h e eye. T h e m i c r o s c o p e
O p i n i o n s are very divided on this subject: as a
t u b e is p a s s e d t h r o u g h t h e c i r c u l a r a p e r t u r e .
r u l e , t h e m a j o r i t y of t h e o r e t i c i a n s s a y t h a t t h e
T h e groove a c c o m m o d a t e s the nose. W i t h this
left e y e s h o u l d b e u s e d for o b s e r v a t i o n . T h e
v e r y s i m p l e t y p e of s h i e l d , t h e d r a w i n g a p p a r -
r e a s o n g i v e n for this p r e f e r e n c e is t h a t t h e r i g h t
a t u s c a n b e u s e d , s i n c e all of o n e side of t h e
eye m u s t r e m a i n free for d r a w i n g , w i t h o r
m i c r o s c o p e r e m a i n s free.
w i t h o u t r o o m l i g h t . I n p r a c t i c e , t h i s r u l e is
Stiassnie m a n u f a c t u r e a d o u b l e shield covered
r a r e l y o b s e r v e d . I n t h e first p l a c e , m a n y p e o p l e
with a green plush. This a p p a r a t u s , designed by
find g r e a t difficulty in m a k i n g m i c r o s c o p i c
o b s e r v a t i o n s w i t h t h e left eye. R a t h e r t h a n b e i n g D r E t . S e r g e n t , is v e r y e a s y to u s e a n d e n a b l e s
forced to r e t r a i n o n e ' s e y e s , w h i c h is p a r t i c u l a r l y o b s e r v a t i o n s to b e m a d e w i t h e i t h e r e y e . I t s o n l y
difficult a t t h e b e g i n n i n g of m i c r o s c o p i c s t u d i e s , d r a w b a c k is t h a t it p r e v e n t s t h e d r a w i n g a p p a r -
it is p r e f e r a b l e to m a k e o b s e r v a t i o n s w i t h t h e a t u s from b e i n g u s e d , b e c a u s e it o c c u p i e s b o t h
right eye. O b s e r v a t i o n s can be carried out s i d e s of t h e m i c r o s c o p e a t t h e s a m e t i m e . I t is
e q u a l l y well w i t h e i t h e r e y e , d e p e n d i n g o n p e r - a t t a c h e d to the microscope tube by a ring.
sonal preference. T h o s e w h o can work alternate- T h e s e eyeshields m u s t be completed by addi-
ly w i t h e i t h e r eye h a v e a b i g a d v a n t a g e w h i c h t i o n of a l a r g e s c r e e n of b l a c k e n e d c a r d b o a r d
e n a b l e s t h e m to a v o i d t h e fatigue of m o n o c u l a r m o u n t e d o n a s u p p o r t , to p r o v i d e sufficient
observation. p r o t e c t i o n a g a i n s t e x t r a n e o u s light. O n e v e r y
s i m p l e m e t h o d of m a k i n g t h e s e s c r e e n s consists
S H O U L D O N E EYE BE CLOSED? of t a k i n g a s m a l l h o l l o w i r o n s t a n d a n d fixing a
W e a g r e e w i t h m o s t a u t h o r s t h a t b o t h eyes l a r g e s h e e t of b l a c k c a r d b o a r d o n to t h e r i n g s
should be open when making microscope w i t h a b r a s s w i r e . C a r e m u s t b e t a k e n to r o t a t e
o b s e r v a t i o n s . I t is v e r y t i r i n g to k e e p o n e eye t h e r i n g s v e r t i c a l l y . T h e h e i g h t of t h e s c r e e n c a n
closed for a l o n g t i m e . M u s c u l a r c o n t r a c t i o n a n d b e a d j u s t e d a s r e q u i r e d b y m o v i n g t h e rings
pressure on the eyeball both quickly p r o d u c e a l o n g t h e v e r t i c a l shaft of t h e s t a n d . T h e u s e of
fatigue a n d e v e n e y e s t r a i n o r h e a d a c h e . I t is t h i s t y p e of s c r e e n is g r e a t l y r e c o m m e n d e d ,
20 Instruments and techniques

e s p e c i a l l y w h e n w o r k i n g w i t h s t r o n g artificial p o s s i b l e . T h u s t h e r e d u c t i o n of light to t h e
l i g h t i n g . U s e of t h e d r a w i n g a p p a r a t u s e n a b l e s r e t i n a c a u s e d b y t h i s reflex a c t i o n d o e s n o t c o m e
e a s y r e g u l a t i o n of t h e a m o u n t of light falling o n into play.
t h e p a p e r . S c r e e n s of this t y p e w i t h c u r v e d A. A r n u l f designed a revolving eyepiece which
surfaces are available commercially. e n l a r g e s t h e exit p u p i l to o v e r c o m e this i n c o n -
v e n i e n c e . F i n a l l y , a n o t h e r s o l u t i o n is to u s e a
BINOCULAR OBSERVATION m o s a i c of m i c r o l e n s e s f u n c t i o n i n g a s a p r o j e c -
I n binocular observation the interocular dis- t i o n s c r e e n a n d r e v o l v i n g r a p i d l y . T h i s gives
t a n c e m u s t b e a d j u s t e d c o r r e c t l y to m e r g e t h e e x c e l l e n t r e s u l t s , a n d o b s e r v a t i o n is m o r e c o m -
two images completely. T h e cornea must be f o r t a b l e b e c a u s e t h e p o s i t i o n of t h e eye is m u c h
close e n o u g h to t h e e y e p i e c e so t h a t t h e w h o l e less c r i t i c a l . T h i s a r r a n g e m e n t c a n b e f u r t h e r
field of vision c i r c u m s c r i b e d b y t h e a p e r t u r e i m p r o v e d b y a l l o w i n g s t e r e o s c o p i c vision w i t h a
d i a p h r a g m c a n b e seen a t t h e s a m e t i m e . By single objective i n s t r u m e n t .
a l t e r n a t e l y closing o n e eye a n d t h e n t h e o t h e r , O n e c a n a c h i e v e s t e r e o s c o p i c vision o n a
p o s s i b l e differences of vision b e t w e e n t h e t w o screen with a two-objective i n s t r u m e n t by using
eyes c a n b e easily c o r r e c t e d b y a d j u s t i n g t h e a suitable placed Fresnel lens.
r i n g o n o n e of t h e t u b e s . If t h e o p e r a t o r suffers
from a s t i g m a t i s m , s p e c t a c l e s m u s t still b e w o r n , O P E R A T I N G T H E FINE A D J U S T M E N T
a l t h o u g h one m u s t check t h a t the eyepieces used It has already been stressed that d u r i n g micro-
h a v e a n exit p u p i l far e n o u g h a w a y from t h e s c o p e o b s e r v a t i o n o n e m u s t a d j u s t t h e fine
e y e p i e c e to e n s u r e t h a t t h e glass of t h e s p e c t a c l e s a d j u s t m e n t c o n t i n u a l l y w i t h t h e right h a n d . T h e
d o e s n o t s c r a t c h t h e e y e p i e c e of t h e m i c r o s c o p e . m i c r o s c o p e c a n d i s p l a y o n l y o n e p l a n e of t h e
I f o n e c a n n o t a v o i d t h e s p e c t a c l e lens c o m i n g o b j e c t clearly a n d , b e c a u s e of t h e c u r v a t u r e of
into contact with the eyepieces or one c a n n o t t h e field, o n l y p a r t of this p l a n e c a n b e seen in
replace the eyepieces, the spectacles can be s h a r p detail at the s a m e time. Small objects act
m o u n t e d i m m o v a b l y o n to t h e c a p s c o v e r i n g a s t h r e e - d i m e n s i o n a l solid b o d i e s , a n d s e c t i o n s
e a c h e y e p i e c e . I n this c a s e , if t h e s p e c t a c l e s h a v e definite t h i c k n e s s e s . T h u s t h e s h a p e a n d
c o r r e c t a definite a s t i g m a t i s m , a line i n d i c a t i n g p r o p o r t i o n s of t h e s e o b j e c t s c a n o n l y b e s t u d i e d
the sagittal plane m u s t be m a r k e d on the specta- b y m e n t a l l y s u p e r i m p o s i n g serial o p t i c a l sec-
cle l e n s e s . t i o n s t h a t h a v e b e e n e x a m i n e d b y successively
T h e preparation should not be moved con- t u r n i n g t h e fine a d j u s t m e n t .
t i n u o u s l y for r a p i d e x a m i n a t i o n , b u t s t e p b y S h a r p n e s s of o b s e r v a t i o n is a c h i e v e d b y a
s t e p , e a c h s t e p b e i n g e q u i v a l e n t to t w o - t h i r d s of c o m b i n a t i o n of t h e focusing of t h e o p t i c a l s y s t e m
t h e d i a m e t e r of t h e field. T h e o b s e r v e r m u s t a n d t h e a c c o m m o d a t i n g p o w e r of t h e eye. T h e
o b s e r v e o n l y t h e c e n t r e of e a c h field a t rest, after latter m u s t not on any account be brought into
t r a i n i n g his o w n p e r i p h e r a l vision. T h i s t r a i n i n g p l a y in m i c r o s c o p i c o b s e r v a t i o n s . If focusing is
c a n be accomplished by practising speed- insufficient a n d if t h e eye tries to c o m p e n s a t e
reading, attaining a b o u t 6000 words per m i n u t e . reflexly b y a l t e r i n g t h e c u r v a t u r e of t h e l e n s , t h e
I n o r d e r to a t t a i n s u c h a s p e e d , t h e eye n e e d s to a c c o m m o d a t i n g m u s c l e s fatigue r a p i d l y . O n t h e
b e a b l e to e x a m i n e t h e w o r d s o n a line w h i l e o t h e r h a n d , b y c o n t i n u a l l y a d j u s t i n g t h e focus
following it a n d also to b e a b l e to u n d e r s t a n d t h e w i t h t h e fine a d j u s t m e n t , t h e eye f u n c t i o n s a s
e n t i r e line a t o n e - g l a n c e . T r a i n i n g in p e r i p h e r a l t h o u g h f o c u s i n g o n infinity. T h u s a s h a r p i m a g e
vision also offers o t h e r a d v a n t a g e s : t h i s p a r t of of t h e o b j e c t is f o r m e d o n t h e r e t i n a w i t h o u t t h e
t h e r e t i n a is m o r e sensitive to w e a k i l l u m i n a t i o n accommodating mechanism being brought into
a n d t h e i l l u m i n a t i o n of t h e m i c r o s c o p e c a n b e action.
r e d u c e d ; also, t h e d a z z l e o c c u r r i n g w h e n p a s -
s i n g from a n e m p t y field to a field c o n t a i n i n g a n O P E R A T I O N OF T H E M O V A B L E STAGE
o b j e c t is l e s s e n e d . After a d j u s t i n g t h e i l l u m i n a t i o n b y m e a n s of t h e
I t s h o u l d b e e m p h a s i z e d t h a t t h e eye a t t h e r a t c h e t a n d i r i s - d i a p h r a g m , t h e left h a n d of t h e
m i c r o s c o p e is m o r e easily d a z z l e d t h a n it is in observer displaces the preparation with the dual
ambient daylight illumination, because at high c a r r i a g e of t h e m o v a b l e s t a g e . T h e p r e p a r a t i o n
p o w e r t h e b e a m of light a t t h e exit p u p i l of t h e is d i s p l a c e d slightly b y b a c k w a r d a n d f o r w a r d
e y e p i e c e h a s a s m a l l d i a m e t e r , a n d t h e iris m o v e m e n t s : e a c h p o r t i o n is s t u d i e d a l o n g its
c a n n o t s t o p it d o w n , e v e n w h e n closed a s far as w h o l e w i d t h b y l a t e r a l d i s p l a c e m e n t (Figure
Basic instruments 21
Conditions of m i c r o s c o p i c v i s i o n
I t is n e c e s s a r y to u n d e r s t a n d t h e n a t u r e of
m i c r o s c o p i c v i s i o n , to i n t e r p r e t t h e i m a g e
f o r m e d b y a m i c r o s c o p e c o r r e c t l y . M a n y diffi-
c u l t i e s a n d e r r o r s will b e a v o i d e d if it is clearly
u n d e r s t o o d t h a t it is a s p e c i a l t y p e of v i s i o n a
Figure 1.27. Procedure for the systematic
examination of a preparation m e t h o d of p e r c e i v i n g o b j e c t s w h i c h is w h o l l y
different from t h a t to w h i c h w e a r e a c c u s t o m e d .
1.27). If o n e d o e s n o t h a v e a m o v a b l e s t a g e , o n e N o r m a l vision is b i n o c u l a r a n d gives t h e s e n s a -
m u s t g r a s p t h e p r e p a r a t i o n a l o n g its t w o l o n g e r t i o n of d i s t a n c e a n d d e p t h . T h i s s e n s a t i o n is d u e
e d g e s b e t w e e n t h e t h u m b a n d i n d e x finger, w i t h t o t h e s u p e r i m p o s i t i o n of t h e t w o r e t i n a l i m a g e s
t h e m i d d l e finger r e s t i n g o n t h e s t a g e . T h e a n d to t h e m e r g i n g of t h e t w o c o r r e s p o n d i n g
p r e p a r a t i o n t h u s slides w i t h o u t excessive j e r k - perceptions. T h e s e two images are not identical;
ing, a n d the whole section can be e x a m i n e d . t h e y differ to a n i n c r e a s i n g e x t e n t as t h e o b j e c t is
b r o u g h t m o r e closely to t h e eyes. T h e s e n s a t i o n
of d e p t h d i m i n i s h e s w i t h i n c r e a s i n g d i s t a n c e of
Choice of objectives and e y e p i e c e s t h e o b j e c t v i e w e d from t h e o b s e r v e r . O n t h e
A p r e p a r a t i o n s h o u l d a l w a y s b e e x a m i n e d first o t h e r h a n d , m i c r o s c o p e vision is m o n o c u l a r ,
at low p o w e r a n d then at progressively higher e v e n if t h e t u b e is b i n o c u l a r . T h e r e f o r e , w h e n
p o w e r m a g n i f i c a t i o n s . T h e field d i m i n i s h e s a n d looking d o w n a microscope, the original training
t h e t r a n s p a r e n c y d e c r e a s e s in p r o p o r t i o n to t h e of t h e eye m u s t b e i g n o r e d a n d t h e t h i c k n e s s a n d
i n c r e a s e in m a g n i f i c a t i o n . M a g n i f i c a t i o n is d e p t h of t h e o b j e c t m u s t b e e s t i m a t e d b y o t h e r
o b t a i n e d b y u s i n g objectives of i n c r e a s i n g l y methods.
h i g h e r p o w e r s ; in p r i n c i p l e , o n e s h o u l d n o t t r y I n n o r m a l vision, o b j e c t s l o c a t e d a t different
t o o b t a i n it w i t h t h e e y e p i e c e s . T h e e y e p i e c e d i s t a n c e s from t h e o b s e r v e r c a n b e seen clearly
only enlarges the i m a g e supplied by the objec- o n e after a n o t h e r , b u t n o t s i m u l t a n e o u s l y , b y
tive: t h e m o r e p o w e r f u l t h e e y e p i e c e , t h e g r e a t e r m e a n s of t h e a c c o m m o d a t i n g m e c h a n i s m . I n
t h e loss of i m a g e s h a r p n e s s , t r a n s p a r e n c y a n d m i c r o s c o p i c v i s i o n , a c c o m m o d a t i o n is n o t
size. T h e r e f o r e , for r o u t i n e w o r k , low a n d b r o u g h t i n t o p l a y . O n l y o n e v e r y t h i n p l a n e of
m e d i u m p o w e r e y e p i e c e s o n l y s h o u l d b e u s e d ; if the object u n d e r examination can be observed at
g r e a t e r m a g n i f i c a t i o n is n e e d e d , this s h o u l d b e one time.
obtained with m o r e powerful objectives. H i g h O n e o t h e r i m p o r t a n t difference between
p o w e r e y e p i e c e s a r e r e s e r v e d for s p e c i a l c a s e s ; o r d i n a r y vision a n d m i c r o s c o p i c vision r e s u l t s
t h e y r e d u c e definition a n d d o n o t i n c r e a s e r e - from t h e n a t u r e of t h e i l l u m i n a t i o n . T h e o b j e c t s
s o l u t i o n . A s n o t e d p r e v i o u s l y , t h e size of t h e s u r r o u n d i n g u s a r e s e e n b y reflection, d u e to t h e
i m a g e d u e to a n o p t i c a l c o m b i n a t i o n is n o t l i g h t t h a t t h e y diffuse. I n t h e c a s e of t h e m i c r o -
i n d i c a t i v e of its v a l u e w i t h r e g a r d to r e s o l v i n g s c o p e , t h e o v e r w h e l m i n g m a j o r i t y of p r e p a r a -
p o w e r a n d definition. t i o n s is e x a m i n e d in t r a n s m i t t e d light, i.e. b y
t r a n s p a r e n c y . T h e r e s u l t is t h a t refraction p h e -
n o m e n a p l a y a m u c h m o r e i m p o r t a n t role t h a n
w i t h observation with the naked eye. Microsco-
Microscopic observation pic images m u s t be divided into two groups on
t h i s b a s i s . O n e g r o u p c o n s i s t s of i m a g e s f o r m e d
I n the preceding sections we have dealt with the b y s t a i n e d p r e p a r a t i o n s , w h i c h a b s o r b light;
d e s i g n of t h e m i c r o s c o p e a n d t h e m e t h o d s for t h e s e i m a g e s o n l y s h o w t h e c o l o u r a n d o u t l i n e of
u s i n g its i n d i v i d u a l c o m p o n e n t s . S o m e a u t h o r s t h e o b j e c t s . T h e o t h e r g r o u p of i m a g e s is f o r m e d
a s s e r t t h a t t h e r e c a n b e n o fixed r u l e s for u s i n g b y c o l o u r l e s s p r e p a r a t i o n s ; this is t h e c a s e for
m i c r o s c o p e s a n d t h a t t h e m e t h o d of a r r a n g i n g o r g a n i s m s e x a m i n e d in t h e i r n a t u r a l s t a t e w i t h -
t h e i l l u m i n a t i o n a n d of e v a l u a t i n g d e t a i l s of out staining. T h e s e preparations act on the
p r e p a r a t i o n can only be learned by experience. b e a m of light b y diffraction a n d refraction; t h e y
T h e p r e s e n t a u t h o r s d o n o t s h a r e this o p i n i o n . I t s h o w n o t o n l y t h e o u t l i n e of t h e o b j e c t s b u t also
s e e m s to u s to b e i m p o r t a n t to set o u t t h e m o s t t h e d e t a i l s of t h e i r s t r u c t u r e , b e c a u s e of differ-
precise rules possible, based on the physical e n c e s of r e f r a c t i o n o r c o n t r a s t s of light a n d
p r o p e r t i e s of t h e c o m p o n e n t s of t h e m i c r o s c o p e . shade.
22 Instruments and techniques

M i c r o s c o p i c vision also differs from n o r m a l Air bubble


vision o n a c c o u n t of t h e c o n s i d e r a b l e a p e r t u r e of
t h e c o n e of light w h i c h i l l u m i n a t e s t h e p r e p a r a -
t i o n s . T h e d i m e n s i o n s of this l u m i n o u s c o n e
g r e a t l y influence t h e n a t u r e of t h e r e f r a c t i o n .
F i n a l l y , m i c r o s c o p i c vision differs f u n d a m e n t a l - (a) (b) (c)
ly from n o r m a l vision in t h e n a t u r e of t h e Lower Medium Upper
m e d i u m in w h i c h t h e objects a r e c o n t a i n e d . I n
g e n e r a l , w e see a r o u n d u s o b j e c t s s u r r o u n d e d b y
air; in m i c r o s c o p y , o n t h e o t h e r h a n d , t h e
p r e p a r a t i o n s m u s t n e a r l y a l w a y s b e m o u n t e d in
a l i q u i d m e d i u m . I n d e e d , m o u n t i n g in a i r is
g e n e r a l l y i m p o s s i b l e b e c a u s e of d r y i n g a n d (d) (e) (f)
h a r d e n i n g w h i c h a c c o m p a n y it; a l s o , t h e i n t r o - Oil drop

d u c t i o n of a i r i n t o t h e i n t e r s t i c e s of t h e o b j e c t s F i g u r e 1.28. Images of air bubbles and oil


u n d e r e x a m i n a t i o n would p r o d u c e refraction droplets in water at three focal planes: lower,
w h i c h w o u l d m a k e it i m p o s s i b l e to d i s t i n g u i s h medium and upper
fine s t r u c t u r a l f e a t u r e s .
T h e a p p e a r a n c e of t h e o b j e c t s v a r i e s g r e a t l y is seen c l e a r l y (Figure 1.28b). U n d e r t h e s e c o n d i -
a c c o r d i n g to t h e refractive i n d e x of t h e m e d i u m . t i o n s , t h e c e n t r e a p p e a r s a s a v e r y b r i g h t circle
A n o b j e c t w h i c h h a s t h e s a m e refractive i n d e x as s u r r o u n d e d b y a g r e y i s h z o n e o u t s i d e w h i c h lies
t h e m e d i u m in w h i c h it is i m m e r s e d b e c o m e s a l a r g e b l a c k r i n g w i t h s t r i p e s f o r m e d from
i n v i s i b l e . If t h e object h a s a h i g h e r refractive c o n c e n t r i c b r i g h t t h i n r i n g s , c o r r e s p o n d i n g to
i n d e x t h a n t h e m e d i u m , it t a k e s o n t h e c h a r a c - diffraction fringes.
teristics of a solid b o d y ; finally, if t h e o b j e c t h a s I f t h e o b j e c t i v e is l o w e r e d to focus o n t h e
a l o w e r refractive i n d e x t h a n t h e m e d i u m , it l o w e r p a r t of t h e b u b b l e , t h e c e n t r a l w h i t e circle
a p p e a r s as a hollow body. Ranvier p u t forward b e c o m e s s m a l l e r a n d b r i g h t e r ; it is also m o r e
a n e x c e l l e n t e x a m p l e to i l l u s t r a t e t h e s e p h e - c l e a r l y defined a n d loses its g r e y b o r d e r . T h e
n o m e n a . T h r e e identical bottles are taken a n d b l a c k r i n g s u r r o u n d i n g it is l a r g e r , o u t of focus,
filled w i t h w a t e r , c a r b o n d i s u l p h i d e a n d C a n a d a a n d hazy at the edge, a n d there are several
b a l s a m , r e s p e c t i v e l y . A glass r o d is p l u n g e d i n t o diffraction r i n g s a t t h e p e r i p h e r y (Figure 1.28a).
e a c h of t h e s e t h r e e b o t t l e s . I n t h e first, t h e r o d I f o n e focuses o n t h e u p p e r p a r t of t h e b u b b l e ,
a p p e a r s solid b e c a u s e glass h a s a h i g h e r refrac- t h e c e n t r a l w h i t e circle i n c r e a s e s a n d s p r e a d s .
tive i n d e x t h a n w a t e r . I n t h e s e c o n d , it a p p e a r s A t t h e s a m e t i m e , l a r g e g r e y circles a p p e a r
h o l l o w b e c a u s e glass h a s a l o w e r refractive i n d e x a r o u n d it a n d t h e s e in t u r n a r e s u r r o u n d e d b y a
t h a n c a r b o n d i s u l p h i d e . F i n a l l y , in t h e t h i r d , it b l a c k r i n g m u c h n a r r o w e r t h a n before. T h i s
d i s a p p e a r s because C a n a d a b a l s a m a n d glass b l a c k r i n g is c r o s s e d a t its o u t e r e d g e b y n u m e r -
h a v e t h e s a m e refractive i n d e x . o u s diffraction circles (Figure 1.28c).
W e s h a l l e x a m i n e t w o o b j e c t s of well-defined T h e s e observations are explained by total
s h a p e a n d o p t i c a l p r o p e r t i e s w h i c h often o c c u r i n t e r n a l reflection ( e x c e p t for t h e diffraction
in m i c r o s c o p i c p r e p a r a t i o n s , in o r d e r to a r r i v e a t r i n g s ) . T h e b e a m of l i g h t p a s s i n g t h r o u g h t h e a i r
a b e t t e r u n d e r s t a n d i n g of t h e p h e n o m e n a w h i c h b u b b l e m a y b e c o n s i d e r e d a s c o n s i s t i n g of p a r a l -
t a k e p l a c e in m i c r o s c o p i c vision; t h e y a r e a i r lel r a y s . O f t h e s e , t h e o n l y r a y s w h i c h e n t e r t h e
b u b b l e s a n d fat g l o b u l e s . m i c r o s c o p e a r e t h o s e w h i c h form a n a n g l e w i t h
t h e axis after r e f r a c t i o n , s u c h t h a t its v a l u e d o e s
n o t e x c e e d t h e critical a n g l e for t h e p a s s a g e from
AIR BUBBLES IN WATER w a t e r to a i r (48) o r h a l f t h e a n g l e of t h e
A few a i r b u b b l e s a r e t r a p p e d b e t w e e n a slide a p e r t u r e of t h e o b j e c t i v e , r e l a t i v e to t h e w a t e r .
a n d c o v e r - s l i p in a d r o p l e t of w a t e r o r s a l i v a . A All o t h e r light r a y s u n d e r g o t o t a l reflection; t h i s
b u b b l e is c h o s e n w h i c h is s m a l l e n o u g h n o t to b e m a k e s t h e e d g e s of t h e a i r b u b b l e a p p e a r i n d i s -
c o m p r e s s e d a n d to r e t a i n its s p h e r i c a l s h a p e . t i n c t . Figure 1.29 i l l u s t r a t e s this a n d also e x p l a i n s
T h e condenser m u s t be stopped d o w n to a small t h e v a r i a t i o n s in d i m e n s i o n of t h e i n d i s t i n c t
d i a m e t e r (Figure 1.28). F i r s t , t h e o b j e c t i v e is r i n g . O n l y r a y s a' a n d a" p e n e t r a t e t h e o b j e c -
focused so t h a t t h e c i r c u m f e r e n c e of t h e b u b b l e tive, s i n c e t h e a p e r t u r e is n o t sufficiently l a r g e to
Basic instruments 23

b u b b l e . T h e rings become m o r e distinct because


t h e a n g l e of a p e r t u r e of t h e i l l u m i n a t i n g b e a m
decreases.

A I R B U B B L E S IN C A N A D A BALSAM
A i r b u b b l e s c a n b e t r a p p e d in C a n a d a b a l s a m
s i m p l y b y s t i r r i n g it w i t h a s m a l l glass r o d . T h e
c r i t i c a l a n g l e is k n o w n to b e s m a l l e r b e c a u s e t h e
difference b e t w e e n t h e refractive i n d i c e s of t h e
t w o m e d i a is g r e a t e r . C o n s e q u e n t l y in C a n a d a
b a l s a m , w h i c h h a s a m u c h h i g h e r refractive
i n d e x t h a n w a t e r , o n l y t h e r a y s falling close t o
t h e p o l e of t h e a i r b u b b l e r e a c h t h e o b s e r v e r ' s
eye.

F A T G L O B U L E S IN W A T E R
a a' a " a' a " "
A d r o p of oil is p l a c e d in a flask c o n t a i n i n g
F i g u r e 1.29. Light path in an air bubble examined in water a b o u t 10 m l of w a t e r a n d s h a k e n . A d r o p of this
e m u l s i o n is e x a m i n e d b e t w e e n a slide a n d cover-
s l i p . C a r e m u s t b e t a k e n to c h o o s e a g l o b u l e
r e c e i v e r a y a'". R a y a"" is totally reflected
w h i c h is s m a l l e n o u g h n o t t o b e d e f o r m e d b y t h e
b e c a u s e its a n g l e of i n c i d e n c e e x c e e d s t h e c r i t i c -
p r e s s u r e . A h a z y , g r e y i s h m a s s is p e r c e i v e d
a l a n g l e . N o w , if t h e o b j e c t i v e is focused o n
w h e n f o c u s i n g o n t h e l o w e r p a r t of t h e g l o b u l e
p l a n e xy, p a s s i n g t h r o u g h t h e e q u a t o r of t h e a i r
(Figure 1.28d), w h i c h s h o w s t h r e e c o n c e n t r i c
b u b b l e , t h e c e n t r a l circle of a i r w o u l d h a v e a
zones: a very bright outer zone, a darker middle
d i a m e t e r of Ca"?, s i n c e t h e eye p e r c e i v e s t h e r a y
z o n e a n d a g r e y c e n t r a l z o n e . W h e n focusing o n
a" in t h e axis a l o n g w h i c h it is r e f r a c t e d . O n t h e
t h e c e n t r e of t h e g l o b u l e , t h e l a t t e r is seen to b e
o t h e r h a n d , if o n e focuses o n t h e l o w e r p a r t of
b o u n d e d by a sharp, black, n a r r o w ring with
the bubble, one only observes a bright n a r r o w
s t r i p e s c o n s i s t i n g of diffraction circles. T h e rest
circle h a v i n g a d i a m e t e r o f C I U ' V F i n a l l y , w h e n
of t h e g l o b u l e a p p e a r s b r i g h t g r e y (Figure 1.28e).
o n e focuses o n t h e u p p e r p a r t of t h e b u b b l e , t h e
M F i n a l l y , w h e n focusing o n t h e u p p e r p a r t of t h e
b r i g h t circle b e c o m e s m u c h l a r g e r ( C 2 3 ) . F o r
g l o b u l e , t h e b l a c k ring is s e e n t o e x p a n d a n d
e a c h of t h e s e p o s i t i o n s of t h e o b j e c t i v e , t h e
b e c o m e h a z y a n d g r e y i s h a t its p e r i p h e r y . A
i n d i s t i n c t z o n e c o r r e s p o n d s to t h e p a r t of t h e
v e r y b r i g h t a n d s h a r p l y defined circle c o m e s
b u b b l e l o c a t e d o n t h e o u t s i d e of r a y a". I n
i n t o v i e w a t t h e c e n t r e (Figure 1.28/).
s u m m a r y , t h e b u b b l e of a i r a c t s a s b i c o n c a v e
lens a n d c h a n g e s t h e p a r a l l e l b e a m t o a d i v e r - T h e s e p h e n o m e n a a r e e x a c t l y t h e o p p o s i t e of
gent one. t h o s e o b s e r v e d in a i r b u b b l e s in w a t e r o r in
b a l s a m : t h e i n d i s t i n c t z o n e d i m i n i s h e s i n s t e a d of
C o m p a r i n g Figures 1.28 a n d 1.29, t h e s e t h e o r e -
i n c r e a s i n g w h e n t h e objective is l o w e r e d g r a -
tical c o n s i d e r a t i o n s c o r r e s p o n d a l m o s t e x a c t l y
dually.
to t h e r e s u l t s o b t a i n e d in p r a c t i c e . I n fact, t h e
e d g e of t h e a i r b u b b l e is f o u n d n o t t o b e
c o m p l e t e l y i n d i s t i n c t ; this is d u e t o t h e fact t h a t Practical applications
rays, u n d e r g o i n g refraction too great to enable
t h e m to p e n e t r a t e the objective, are partly re- REFRACTION
flected i n t o t h e i n t e r i o r of t h e b u b b l e a n d a r e T h e s e three experiments help one to recognize
t h u s p a r t l y o b s e r v e d b y t h e e y e of t h e o b s e r v e r . a i r b u b b l e s a n d fatty g l o b u l e s a n d n o t t o m i s -
I n c o n c l u s i o n , t h e n u m e r o u s diffraction rings t a k e t h e m for h i s t o l o g i c a l s t r u c t u r e s . T h e y also
a r e f o r m e d a s a r e s u l t of t h e i n t e r a c t i o n b e t w e e n d e m o n s t r a t e t h e difference b e t w e e n objects h a v -
t h e i n d i s t i n c t z o n e of t h e b u b b l e a n d t h e r a y s i n g h i g h e r o r l o w e r refractive i n d i c e s t h a n t h a t
a c t i n g a s a d i a p h r a g m p r o d u c i n g diffraction of t h e m e d i u m in w h i c h t h e y a r e i m m e r s e d .
fringes. T h e s e r i n g s b e c o m e m o r e n u m e r o u s a s O b j e c t s w i t h a h i g h e r refractive i n d e x t h a n
t h e ' d i a p h r a g m ' , i.e. t h e b r i g h t circle, d e c r e a s e s t h e m e d i u m , s u c h a s oil in w a t e r , s h o w a c e n t r a l
in size. T h e r e f o r e , t h e n u m b e r of rings i n c r e a s e s z o n e w h i c h i n c r e a s e s in b r i g h t n e s s a n d b e c o m e s
a s o n e focuses o n t h e d e e p e r p o r t i o n of t h e narrower, surrounded by an indistinct ring
24 Instruments and techniques

w h i c h i n c r e a s e s in size as t h e p o s i t i o n of t h e F i g u r e 1.30. Determination


p o i n t e x a m i n e d a p p r o a c h e s t h e u p p e r surface. of the refractive index of a
droplet by immersion in a
T h e y act as biconvex lenses. O n the other h a n d ,
liquid of known refractive
o b j e c t s w i t h a l o w e r refractive i n d e x t h a n t h e index with oblique
m e d i u m , s u c h as a i r b u b b l e s in w a t e r , a r e illumination. The dark
s u r r o u n d e d by an indistinct dark ring, which border disappears when the
i n c r e a s e s in size as t h e p o s i t i o n of t h e p o i n t refractive indices are equal;
it is on the side of the screen
e x a m i n e d a p p r o a c h e s t h e l o w e r surface of t h e if the refractive index of the
o b j e c t . T h i s effect is t h e m o r e a c c e n t u a t e d t h e droplet is greater than that
g r e a t e r t h e difference b e t w e e n t h e t w o refractive of the medium, and on the
i n d i c e s ; for e x a m p l e , w h e t h e r t h e a i r b u b b l e is in other side if it is less
w a t e r o r b a l s a m . S u c h objects a c t as b i c o n c a v e
lenses. d i r e c t i o n a s t h e b e a m of light, w h e r e a s if it h a s a
O n the other h a n d , convex objects with high- l o w e r refractive i n d e x t h e y d i v e r g e in o p p o s i t e
e r refractive i n d i c e s t h a n t h e m e d i u m a c t like d i r e c t i o n s . A s t h e i m a g e s a r e i n v e r t e d in t h e
fatty g l o b u l e s in w a t e r , w h i l e c o n c a v e o b j e c t s m i c r o s c o p e , t h e following p o i n t s a r e n o t i c e d : t h e
w i t h l o w e r refractive i n d i c e s t h a n t h e m e d i u m s h a d o w e x t e n d s o v e r t h e field of t h e m i c r o s c o p e
a c t like a i r b u b b l e s in w a t e r . o n t h e s i d e of t h e finger o r p i e c e of c a r d b o a r d ; if
T h i s t e c h n i q u e c a n b e u s e d to e s t i m a t e t h e it is a r r a n g e d so t h a t a b o u t h a l f t h e field is
refractive i n d e x of a s u b s t a n c e , p r o v i d e d t h a t it d a r k e n e d , a s u b s t a n c e w i t h a h i g h e r refractive
d o e s n o t m i x w i t h t h e l i q u i d in w h i c h it is i n d e x t h a n t h e m e d i u m will b e d a r k o n t h e d a r k
e x a m i n e d . A series of l i q u i d s of k n o w n refractive s i d e of t h e field (Figure 1.30) a n d b r i g h t o n t h e
i n d i c e s is p r e p a r e d a n d t h e o b j e c t s a r e i m m e r s e d bright side. O n the other h a n d , a s u b s t a n c e with
in t h e m successively. T h e refractive i n d i c e s of a l o w e r refractive i n d e x t h a n t h e m e d i u m will b e
the objects can be estimated either by finding a b r i g h t o n t h e d a r k side of t h e field a n d d a r k o n
l i q u i d of t h e s a m e i n d e x o r b y r a n k i n g it b e - the bright side. A s u b s t a n c e which has the s a m e
t w e e n t w o l i q u i d s of h i g h e r a n d l o w e r refractive r e f r a c t i v e i n d e x a s t h e m e d i u m will b e s u r -
indices. r o u n d e d b y c o l o u r e d fringes b e c a u s e t h e l i q u i d
T h i s i m m e r s i o n m e t h o d is c o m m o n l y u s e d b y nearly always has a dispersive power greater
mineralogists a n d chemists. As described here, than the substance under examination.
it o n l y a p p l i e s to i s o t r o p i c o r s i m p l e r e f r a c t i n g T h e s e f e a t u r e s o n l y b e c o m e c l e a r if t h e c o n -
s u b s t a n c e s . T h e i n d e x of a n i s o t r o p i c o r d o u b l e d e n s e r is set to its m a x i m u m , t h e iris is c o m -
r e f r a c t i n g s u b s t a n c e s h a s different v a l u e s d e - p l e t e l y o p e n a n d a m e d i u m m a g n i f i c a t i o n of
p e n d i n g on the orientation; a polarizing micro- a b o u t 2 0 0 d i a m e t e r s is u s e d . If t h e c o n d e n s e r is
s c o p e m u s t b e u s e d to d e t e r m i n e it. lowered, the features become inverted and m u c h
I t h a s b e e n seen t h a t differences in refraction less w e l l - d e f i n e d . L i q u i d s of g r a d u a t e d refrac-
a r e o b s e r v e d e v e n in a x i a l light. T h e y b e c o m e tive i n d i c e s c a n b e p r e p a r e d u s i n g e i t h e r diffe-
m o r e p r o n o u n c e d u n d e r o b l i q u e light b e c a u s e r e n t p u r e s u b s t a n c e s of k n o w n c o n s t a n t s o r
the dark or luminous b a n d s are then more artificial m i x t u r e s (cf. Table 1.2 in S e c t i o n 1.10).
m a r k e d o n o n e side t h a n o n t h e o t h e r . A t t h e
s a m e time, their position depends on the illumi- DIFFRACTION
n a t i n g b e a m a n d o n t h e difference in refraction Diffraction fringes c a n also c a u s e a r t e f a c t s
b e t w e e n t h e o b j e c t s t u d i e d a n d t h e l i q u i d in w h i c h m a y b e m i s t a k e n b y t h e o b s e r v e r for a
w h i c h it is i m m e r s e d . r e a l s t r u c t u r e . T h e r e a r e s e v e r a l m e t h o d s of
T o o b t a i n o b l i q u e i l l u m i n a t i o n , a p i e c e of r e c o g n i z i n g t h e i r t r u e n a t u r e . T h e y a r e all b a s e d
c a r d b o a r d c u t in a s t r a i g h t line, o r s i m p l y a o n d e t e r m i n i n g w h e t h e r t h e s e fringes c a n b e
finger, is s l i p p e d u n d e r t h e c o n d e n s e r w i t h t h e magnified a n d whether their spacing can be
iris d i a p h r a g m s o m e d i s t a n c e a w a y o r w i d e m o d i f i e d . I f t h e y c a n , this m e a n s t h a t t h e y a r e
o p e n . T h e u s e of o b l i q u e i l l u m i n a t i o n s y s t e m s in diffraction fringes. F o r this p u r p o s e , t h e a p e r -
s o m e s u b - s t a g e s is n o t a d v i s a b l e b e c a u s e it d o e s t u r e m u s t b e r e d u c e d b y closing d o w n t h e
n o t give a n a c c u r a t e i d e a of t h e d i r e c t i o n of t h e d i a p h r a g m or the preparation m u s t be observed
i l l u m i n a t i n g b e a m (Figure 1.30). u n d e r m o n o c h r o m a t i c yellow light. I n t h e first
I f t h e object h a s a h i g h e r refractive i n d e x t h a n c a s e , t h e fringes b e c o m e m o r e d i s t i n c t a n d c o n -
t h e m e d i u m , t h e r a y s c o n v e r g e in t h e s a m e t r a c t e d as t h e d i a p h r a g m b e c o m e s s m a l l e r ; in
Basic instruments 25

t h e s e c o n d c a s e , if o n e uses a s o d i u m l a m p o r , s u r f a c e s b y c a p i l l a r y a c t i o n . I f h e a t is to b e
m o r e s i m p l y , if o n e i n s e r t s b l u e a n d r e d g l a s s a p p l i e d , o n e s h o u l d u s e o n l y r a p i d friction of a
filters in t h e d i a p h r a g m c a r r i e r , t h e diffraction c o r d a r o u n d t h e n e c k of t h e b o t t l e o r flask, s i n c e
fringes c o n t r a c t a n d e x p a n d in t u r n . o t h e r m e t h o d s m a y c a u s e t h e flask to e x p l o d e .
A t t h e b e g i n n i n g of t h i s c h a p t e r , a b a s i c (ix) O n e s h o u l d n e v e r l e a v e a flask o p e n o r
d i s t i n c t i o n w a s m a d e b e t w e e n t h e i m a g e s of unlabelled.
coloured objects or the a m p l i t u d e p r o d u c e d by (x) G l a s s b e a d s s h o u l d b e p l a c e d in a n y flasks
a b s o r p t i o n a n d t h e i m a g e s of c o l o u r l e s s o b j e c t s c o n t a i n i n g r e a g e n t l i a b l e t o o x i d i z e , so t h a t t h e
o r p h a s e i m a g e s d u e to reflection, r e f r a c t i o n a n d l i q u i d r e a c h e s t h e level of t h e n e c k a n d t h e r e is
diffraction. T h e s e l a t t e r p h e n o m e n a a r e ex- little a i r i n s i d e t h e flask.
tremely complicated a n d their interpretation (xi) N o t e s s h o u l d b e m a d e of e v e r y p r o c e d u r e
c a n b e v e r y difficult; t h e light a n d d a r k z o n e s immediately.
a n d t h e diffraction fringes c a n b e m i s t a k e n for (xii) S p e c i a l o r d i s p o s a b l e c o n t a i n e r s o r
m e m b r a n e s o r s t r i a t u m s , , so o n e m u s t b e o n accessories should be used w h e n taking samples
g u a r d against these artefacts. O n the other o r w e i g h i n g t h e m ; to a v o i d c o n t a m i n a t i o n t h e y
h a n d , images formed by absorption are more s h o u l d b e w a s h e d before r e - u s e .
a c c u r a t e a n d give b e t t e r r e p r o d u c t i o n of t h e (xiii) T o x i c p r o d u c t s s h o u l d n o t b e d i s -
o u t l i n e s of t h e o b j e c t s ; t h e r e f o r e , t h e i r i n t e r - c h a r g e d i n t o t h e d r a i n w i t h o u t first r e n d e r i n g
p r e t a t i o n is m u c h less s u b j e c t to e r r o r . them harmless.
(xiv) I n d i v i d u a l p i p e t t e s s h o u l d b e u s e d for
each reagent, a n d they should be washed and
Precautions in a m i c r o s c o p e laboratory dried between successive operations.
(xv) R e s i n s , m o u n t i n g m e d i a , dyestuffs a n d
T h e following r u l e s s h o u l d b e o b s e r v e d , in o r d e r
all r e a g e n t s s e n s i t i v e to light s h o u l d b e s t o r e d in
t o w o r k efficiently a n d c o m f o r t a b l y :
darkness.
(i) A b s o l u t e c l e a n l i n e s s is e s s e n t i a l . (xvi) A c i d s a n d v o l a t i l e p r o d u c t s s h o u l d b e
(ii) I t is a l w a y s d e s i r a b l e to h a v e a p l a n of k e p t in s p e c i a l s t o r e s .
w o r k w i t h e s t i m a t e d t i m e s for v a r i o u s o p e r a - (xvii) O n e s h o u l d n o t b e i m p a t i e n t o r lack
t i o n s , a n d to n o t e in a d v a n c e t h e a m o u n t s of concentration while at work.
r e a g e n t s to b e u s e d . T h o s e t h a t c a n b e k e p t (xviii) O n e s h o u l d n o t e x p e c t e v e r y i n d i -
s h o u l d b e p r e p a r e d b e f o r e h a n d in sufficient v i d u a l e x p e r i m e n t to b e successful, n o r d r a w
quantities. conclusions too rapidly. T h e r e m a y be procedu-
(Hi) Sufficient w o r k surfaces a r e n e c e s s a r y to ral faults, b a d reagents or b a d objects.
carry out procedures with the m i n i m u m dis- (xix) G l a s s w a r e s h o u l d b e w a s h e d w i t h c h r o -
placement. m i c a c i d ( 2 0 g of p o t a s s i u m d i c h r o m a t e + 100 m l
(iv) O n e s h o u l d b e p r o t e c t e d a g a i n s t c o n - o f w a t e r + 100 m l of c o n c e n t r a t e d s u l p h u r i c
t a m i n a t i o n from toxic p r o d u c t s b y f r e q u e n t a c i d ) . T h e r e s h o u l d b e sufficient l i q u i d to i m -
c h a n g e s of l a b o r a t o r y c o a t s , b y w e a r i n g t h i n m e r s e all t h e g l a s s w a r e , a n d o b j e c t s , p a r t i c u l a r -
r u b b e r gloves for h a n d l i n g c o r r o s i v e p r o d u c t s ly slides, s h o u l d n o t b e s t a c k e d .
a n d b y w a s h i n g t h e h a n d s e a c h t i m e after (xx) Slides a n d c o v e r - s l i p s , i n c l u d i n g n e w
h a n d l i n g toxic p r o d u c t s . o n e s , s h o u l d b e w a s h e d before u s e .
(v) S o m e s u b s t a n c e s e m i t toxic v a p o u r s , e.g. (xxi) Slides a n d c o v e r - s l i p s m a y b e ' d r y -
v o l a t i l e a c i d s , d i o x a n e , p h e n o l s , c h l o r i n e a n d its cleaned' by r u b b i n g t h e m with a small a m o u n t
c o m p o u n d s , etc. E x p e r i m e n t s should be carried of m a g n e s i u m u n t i l all a d h e r i n g p o w d e r h a s
o u t in a f u m e c u p b o a r d o r in fresh air. d i s a p p e a r e d . Glass surfaces m a y be m a d e wet-
(vi)' A c i d s s h o u l d b e p o u r e d i n t o t h e i r table, by passing them rapidly through the blue
d i l u e n t , n e v e r vice v e r s a . f l a m e of a B u n s e n b u r n e r .
(vit) V a s e l i n e a p p l i e d to g r o u n d - g l a s s s t o p - (xxii) C u l t u r e s s h o u l d b e t r e a t e d w i t h f o r m a l -
p e r s of w i d e - m o u t h e d b o t t l e s o r flasks e n a b l e s d e h y d e , before t h e y a r e d i s c a r d e d .
t h e m to b e o p e n e d easily. (xxiii) F o r m a l i n a n d p h e n o l h a v e sufficient
(viii) S t o p p e r s m a y b e l o o s e n e d w i t h e t h a n o l , v a p o u r p r e s s u r e for t h e i r v a p o u r s to sterilize
h y d r o g e n p e r o x i d e o r S g u y ' s m i x t u r e (5 m l of small objects, b u t phenol attacks metallic instru-
g l y c e r i n , 5 m l of w a t e r , 10 m l of c h l o r a l h y d r a t e , ments.
5 m l of 2 5 % h y d r o c h l o r i c a c i d ) w h i c h p e n e t r a t e s (xxiv) I n o r d e r to k e e p h y g r o s c o p i c r e a g e n t s
26 Instruments and techniques

such as absolute ethanol, acetone a n d dioxane (b) R e v o l v i n g d i a p h r a g m s modify light in t i m e ;


anhydrous, anhydrous calcium chloride, copper they cannot be used with direct current
sulphate, calcium carbide or metallic sodium a m p l i f i e r s . S h u t t e r filters modify t h e n u m e r -
s h o u l d b e u s e d . T h e last t w o r e a g e n t s a r e ex- ical a p e r t u r e .
t r e m e l y d a n g e r o u s , t h e first b e c a u s e it gives off (c) N e u t r a l g r e y w e d g e s a r e u n d o u b t e d l y t h e
a c e t y l e n e , t h e s e c o n d b e c a u s e it b u r n s o n c o n - b e s t , b u t t h e y a r e difficult to p r o d u c e . If
tact with water. they are obtained photographically, the
(xxv) C h l o r o f o r m o r b e n z e n e c a n b e u s e d t o g r a i n of t h e e m u l s i o n m a y interfere; if t h e y
o p e n flasks c o n t a i n i n g a r e s i n . are obtained by superimposing charged
(xxv) A r u b b e r a i r b u l b is useful to c l e a n g e l a t i n o n I n d i a n ink, t h e y d o n o t l a s t l o n g ;
o b j e c t i v e o r e y e p i e c e lenses; it s h o u l d b e depositing a metal under vacuum should
squeezed energetically. Lens p a p e r or a brush n o t b e u s e d b e c a u s e it c a u s e s i n t e r f e r i n g
s h o u l d o n l y b e u s e d if d u s t r e m a i n s after b l o w - reflections.
i n g . G r e a s e m a r k s s u c h a s fingerprints s h o u l d b e (d) A b s o r b e n t s o l u t i o n s a r e v e r y effective b u t
removed with ethanol and not with hydrocar- m o d e r n microscopes d o not generally pro-
bons. O n e should never immerse an optical v i d e a p o s i t i o n for p l a c i n g a m i c r o c e l l n o r -
s y s t e m in a c l e a n i n g fluid, as t h e r e is risk of m a l to t h e i l l u m i n a t i n g b e a m .
u n g l u i n g the lenses. (e) B e e r - L a m b e r t ' s l a w s t a t e s t h a t t h e a b s o r p -
(xxvii) G a s e o u s h y d r o f l u o r i c a c i d a t t a c k s t i o n of a s o l u t i o n is p r o p o r t i o n a l to its
lenses a n d t a r n i s h e s t h e m , a n d it s h o u l d o n l y b e concentration. T h e same observations ap-
u s e d in fresh a i r . p l y
\
(xxviii) A c e t i c a c i d facilitates u n j a m m i n g of
(f) V a r i a t i o n s in t h e p o w e r s u p p l y c a u s e c o n -
s y r i n g e s a n d n e e d l e s . T h e a c i d is i n t r o d u c e d
s i d e r a b l e v a r i a t i o n in t h e c o l o u r t e m p e r a -
i n t o t h e s y r i n g e s w i t h a fine p i p e t t e a n d i n t o t h e
ture.
needles with a bristle. T h e syringes a n d needles
(g) A l t e r a t i o n s in t h e a p e r t u r e c a n n o t b e m a d e
s h o u l d t h e n b e w a s h e d well a n d g r e a s e d lightly.
w i t h o u t c o n c o m i t a n t c h a n g e s in t h e resolv-
W . R . Goff a d v i s e s i m m e r s i n g j a m m e d s y r i n g e s
ing p o w e r a n d contrast. Therefore, this
in b o i l i n g g l y c e r i n for 30 m i n .
m e t h o d c a n n o t b e u s e d in m o s t c a s e s .
(h) P h o t o s e n s i t i v e filters w h i c h a u t o m a t i c a l l y
Variations in light intensity d a r k e n in l i g h t c a n b e u s e d .
(i) T h e o n l y o p t i c a l l y c o r r e c t m e t h o d for all
I n m i c r o s c o p y t h e light i n t e n s i t y f r e q u e n t l y h a s
c a s e s is t h e u s e of ' z o o m ' m a g n i f i c a t i o n
t o b e a d j u s t e d to e n a b l e t h e eye to w o r k u n d e r
c o n n e c t e d to t h e a p e r t u r e b y a s y s t e m for
o p t i m u m c o n d i t i o n s , o r to c a r r y o u t p h o t o m e t r i c
c o n t i n u o u s focus. M o s t d e s i g n e r s n o w a d a y s
m e a s u r e m e n t s in p h o t o m i c r o g r a p h y o r in s p e c -
use this.
t r o s c o p y . T h e r e a r e e i g h t p r a c t i c a l m e t h o d s of
d o i n g this: I n v i e w of all t h e s e c o n s i d e r a t i o n s , t h e u s e r finds
h i m s e l f r e d u c e d to a m o r e o r less s a t i s f a c t o r y
( 1 ) R e d u c t i o n of p o l a r i z e d light b y r o t a t i n g t h e c o m p r o m i s e . I n p r a c t i c e , o n e of t h e less perfect
polarizer. m e t h o d s u s e d m o s t is t h a t of v a r y i n g t h e p o w e r
(2) U s e of r e v o l v i n g d i a p h r a g m s w i t h v a r i a b l e s u p p l y . I t is h o p e d t h a t d e s i g n e r s in t h e f u t u r e
s e g m e n t s , o r s h u t t e r filters. will i n c o r p o r a t e s u c h efficient m o d i f i c a t i o n s in
(3) U s e of n e u t r a l g r e y w e d g e s of v a r i a b l e their instruments without causing chromatic
density, linear or revolving. imbalance.
(4) P l a c i n g of a b s o r b e n t s o l u t i o n in t h e light
path.
(5) U s e of B e e r - L a m b e r t ' s l a w . Basic m e t h o d s of bright field illumination
(6) V a r y i n g t h e p o w e r s u p p l y to t h e b u l b .
(7) V a r y i n g t h e a p e r t u r e . T h r e e m e t h o d s o n l y a r e in w i d e s p r e a d u s e :
(8) U s i n g a v a r i a b l e focus a r r a n g e m e n t .
(1) D u j a r d i n ' s critical i l l u m i n a t i o n .
These methods are not interchangeable. The (2) K h l e r ' s d o u b l e d i a p h r a g m i l l u m i n a t i o n ,
following p o i n t s a r e r e l e v a n t : the most frequently used.
(3) L o c q u i n ' s bifocal i l l u m i n a t i o n .
(a) R e d u c t i o n of p o l a r i z e d light is n o t s u i t a b l e if
t h e o b j e c t is itself d o u b l e r e f r a c t i n g . O t h e r m e t h o d s m e n t i o n e d in p a s s i n g a r e e i t h e r
Basic instruments 27

v a r i a t i o n s of t h e s e t h r e e , o r else a r e e m p i r i c a l . fringes a t t h e i r e d g e s . O p a l g l a s s , o p a l e s c e n t o n
W e shall consider this later. o n e face o n l y , is r e a d i l y a v a i l a b l e c o m m e r c i a l l y .
I t c a n b e c u t to t h e d i m e n s i o n s of a m i c r o s c o p e
CRITICAL ILLUMINATION slide with a d i a m o n d .
D u j a r d i n , t h e i n v e n t o r of t h e c o n d e n s e r , w h i c h
a t t h a t t i m e w a s c a l l e d a ' c o n c e n t r a t o r ' (a b e t t e r DOUBLE DIAPHRAGM ILLUMINATION
w o r d w h i c h h a s h i s t o r i c a l p r i o r i t y in F r e n c h ) , I n o r d e r to a v o i d t h e pitfalls i n h e r e n t in critical
s t a t e d t h a t t h e q u a l i t y a n d l u m i n o s i t y of t h e i l l u m i n a t i o n m e n t i o n e d a b o v e a n d to u s e s p i r a l
i m a g e s w a s c l e a r l y i m p r o v e d w h e n his c o n - f i l a m e n t b u l b s o r o t h e r m o r e o r less s t r u c t u r e d
d e n s e r p r o j e c t e d t h e i m a g e of his fishtail b u r n e r l i g h t s o u r c e s , K h l e r r e c o m m e n d e d a m e t h o d of
o n t o t h e p l a n e of t h e o b j e c t b e i n g e x a m i n e d ; h e i l l u m i n a t i o n w h i c h c a r r i e s h i s n a m e a n d is
called this m e t h o d 'critical illumination'. T h e u n i v e r s a l l y u s e d . I t m a y e v e n b e s a i d to b e
dislike of m i c r o s c o p i s t s for t h i s m e t h o d d a t e s i n d i s p e n s a b l e for a n y m i c r o s c o p e e q u i p p e d w i t h
from t h e a p p e a r a n c e of s p i r a l f i l a m e n t b u l b s . o b j e c t i v e s of m a g n i f i c a t i o n g r e a t e r t h a n 30 u n -
T h e p r o j e c t i o n of t h e f i l a m e n t in t h e o b j e c t less o n e of t h e o t h e r t w o m e t h o d s is u s e d . I t
p l a n e is c o n f u s i n g , a n d gives rise to d i s t u r b i n g employs double d i a p h r a g m illumination: one
i r r e g u l a r i t i e s in i l l u m i n a t i o n w h i c h a r e e v e n d i a p h r a g m , c a l l e d t h e field d i a p h r a g m , is
m o r e d i s t u r b i n g . A n o t h e r d i s a d v a n t a g e lies in g e n e r a l l y p l a c e d n e a r t h e light s o u r c e , a n d t h e
t h e fact t h a t it is i m p o s s i b l e to l i m i t t h e field o t h e r o n e , c a l l e d t h e s u b - s t a g e d i a p h r a g m , is
i l l u m i n a t e d to t h a t o b s e r v e d . N e v e r t h e l e s s , g e n e r a l l y p l a c e d j u s t b e l o w t h e c o n d e n s e r (Fig-
t h e r e a r e g o o d r e a s o n s for r e v i v i n g t h e p o p u l a r - ure 1.31).
ity of c r i t i c a l i l l u m i n a t i o n for t h e e x a m i n a t i o n of I n p r a c t i c e , t h e i m a g e of t h e field d i a p h r a g m
l a r g e o b j e c t s , b e c a u s e it is t h e o n l y m e t h o d is p r o j e c t e d o n to t h e p l a n e of t h e p r e p a r a t i o n b y
w h i c h gives t r u l y i n c o h e r e n t i l l u m i n a t i o n a t all t h e c o n d e n s e r , w h o s e v e r t i c a l p o s i t i o n is a d -
magnifications. O n e m a y choose a point source j u s t e d t o m a k e t h e i m a g e as s h a r p a s p o s s i b l e .
s u c h a s a h i g h i n t e n s i t y z i r c o n a r c , from w h i c h T h e field d i a p h r a g m r e g u l a t e s t h e i l l u m i n a t i o n
t h e i m a g e of t h e c i r c u l a r s o u r c e w o u l d b e p r o - a p e r t u r e a n d , t h e r e f o r e , t h e c o n t r a s t , t h e field
jected towards the microscope condenser d e p t h a n d the resolving power. T h e s e three
t h r o u g h a v a r i a b l e focus z o o m d e v i c e . T h e variables are not independent: w h e n the di-
c o n d e n s e r d i a p h r a g m r e t a i n s its u s u a l f u n c t i o n a p h r a g m is closed, t h e c o n t r a s t a n d field d e p t h
of r e g u l a t i n g t h e a p e r t u r e . a r e i n c r e a s e d , b u t t h e r e s o l v i n g p o w e r is r e -
T h i s t y p e of i l l u m i n a t i o n c a n a l s o b e o b t a i n e d duced. W h e n starting observations, one should
b y t h e following o r i g i n a l p r o c e d u r e w h i c h gives m a k e c e r t a i n t h a t t h e l a m p f i l a m e n t is well-
v e r y g o o d r e s u l t s . A m i c r o s c o p e slide c o v e r e d b y c e n t r e d w i t h r e s p e c t to t h e c o n d e n s i n g lens a n d
a t h i n l a y e r of p l a n e o p a l glass is u s e d . T h e t h a t t h e b e a m is d i r e c t e d b y t h e m i r r o r i n t o t h e
o b j e c t is m o u n t e d d i r e c t l y o n t h i s o p a l face. T h e c o n d e n s e r , w h i c h m u s t also b e w e l l - c e n t r e d .
p r e p a r a t i o n is i l l u m i n a t e d b y n o r m a l d o u b l e U n f o r t u n a t e l y , in m o s t m i c r o s c o p e s t h e c o n -
d i a p h r a g m i l l u m i n a t i o n , r e g u l a t i n g t h e field d i - d e n s e r has the s a m e design as that sometimes
a p h r a g m in s u c h a w a y t h a t it l i m i t s t h e illu- u s e d in m a g n i f i c a t i o n of t h e o b j e c t i v e ; a r e m o v -
m i n a t i o n of t h e o p a l glass in t h e field o b s e r v e d ; a b l e o r r e t r a c t a b l e lens o n t h e c o n d e n s e r is o n l y
t h e a p e r t u r e d i a p h r a g m of t h e c o n d e n s e r t h e n rarely provided. At high power magnification, a
loses its f u n c t i o n of r e g u l a t i n g t h e a p e r t u r e to v e r y s m a l l p o r t i o n of t h e light is u s e d b e c a u s e
b e c o m e a d i a p h r a g m r e g u l a t i n g t h e i n t e n s i t y of t h e field d i a p h r a g m is n e a r l y closed d o w n , w h i l e
t h e i n c i d e n t l i g h t . T h i s m e t h o d of p r o d u c i n g at low p o w e r magnification the converse occurs.
perfectly i n c o h e r e n t i l l u m i n a t i o n is m u c h s u p e r - Y e t t h e r e is n o d o u b t t h a t m o r e light is n e e d e d
ior to all o t h e r s ; it is t h e o n l y o n e a l l o w i n g a a t h i g h m a g n i f i c a t i o n . I t is p o s s i b l e a s Zeiss
p e r m a n e n t i l l u m i n a t i o n of t h e o b j e c t i v e a t full a n d t h e G a l i l e o L a b o r a t o r y h a v e d o n e for a l o n g
a p e r t u r e w i t h o u t t h e p r o b l e m of s t r a y l i g h t . t i m e t o p r o v i d e a v a r i a b l e focus ( z o o m ) c o n -
H o w e v e r , c a r e m u s t b e t a k e n to e n s u r e t h a t t h e d e n s i n g l e n s in front of t h e field d i a p h r a g m .
o p a l e s c e n t face of t h e g l a s s lies d i r e c t l y a g a i n s t T h i s r e s u l t s in g r e a t e r efficiency a n d , if t h e
t h e object a n d a l s o t h a t it is t h i n . T h i s t y p e of a r r a n g e m e n t is well p l a n n e d , in a n a l m o s t c o n -
i l l u m i n a t i o n is u s u a l l y f o u n d to b e p r e f e r a b l e for s t a n t i l l u m i n a t i o n of t h e i m a g e w h e n t h e objec-
examining large objects; the images are s m o o t h tives a r e c h a n g e d . I t is to b e h o p e d t h a t this
a n d h a v e s h a r p c o n t r a s t , w i t h o u t diffraction s y s t e m will find m o r e w i d e s p r e a d a p p l i c a t i o n .
28

Object plane in w h i c h an image of the object and of


the field diaphragm are located together on t h e
retina of t h e e y e , t h e projection screen or t h e
photographic emulsion

Outlet pupil of t h e microscope w i t h t h e image of


the aperture diaphragm a n d of t h e bulb filament

Eyepiece

Intermediate image of t h e object a n d of t h e field


diaphragm

Front focal plane of the objective w i t h the image of


the aperture diaphragm a n d of t h e bulb filament il
(interference image plane)

Objective

Object observed in t h e image plane of t h e field


diaphragm

Condenser

Inlet pupil of t h e microscope in t h e anterior focal


plane of t h e condenser, aperture diaphragm a n d
image of t h e vertically expanded bulb filament

L a m p condensing
lens

Bulb filament
Field diaphragm

F i g u r e 1.31. Principle of double diaphragm illumination. The image of the bulb filament is axially
enlarged to two level figures; this explains occasional difficulties in obtaining homogeneous illumination of
the field, especially with low power objectives
Basic instruments 29

BIFOCAL I L L U M I N A T I O N power supply have been dispensed with, users


I n bifocal i l l u m i n a t i o n , t h e o p t i c a l s y s t e m of t h e will b e a b l e to d o w i t h o u t costly p h o t o m i c r o -
c o n d e n s e r is d e s i g n e d w i t h a d o u b l e focus, a g r a p h i c a p p a r a t u s , b e c a u s e it will b e p o s s i b l e to
l o n g f o c a l - l e n g t h lens for low p o w e r o b j e c t i v e s , carry out photomicrography with a constant
a n d a short focal-length m i r r o r a n d large aper- e x p o s u r e t i m e , a n d t h e s t a n d will t h e n b e e q u i p -
t u r e for h i g h p o w e r o b j e c t i v e s . T h e c o n d e n s e r is p e d w i t h a single c o l o u r t e m p e r a t u r e - c o r r e c t i n g
fixed o n to a r a t c h e t w h i c h a l l o w s v e r t i c a l filter m o u n t e d rigidly o n it. T h e n t h e r e will b e
d i s p l a c e m e n t b e t w e e n t h e t w o focal p l a n e s . I n n o f u r t h e r n e e d for test p h o t o g r a p h s a n d a u t o -
o r d e r to a d j u s t t h e i l l u m i n a t i o n , t h e o b s e r v e r m a t i c p h o t o g r a p h y a p p a r a t u s . A well-designed
a l t e r s t h e h e i g h t of t h e c o n d e n s e r to o b t a i n t h e a n d well-adjusted microscope works at constant
o p t i m a l image. T h i s image has special c h a r a c - i m a g e l u m i n o s i t y a n d t h i s is e n o u g h to e n s u r e
teristics s i n c e it is f o r m e d b y p e r m a n e n t l y s u p e r - a n e x p o s u r e t i m e of t h e o r d e r of 0.07 s w i t h a
imposing two illuminations: a s h a r p axial a n d 1 0 - 2 0 A S A e m u l s i o n for n e a r l y all o b j e c t s in a
i n t e n s e i l l u m i n a t i o n w h i c h gives c o n t r a s t , a n d a b r i g h t field a n d in w h i t e light. T h e o b s e r v e r will
soft b r o a d i l l u m i n a t i o n w h i c h p r o v i d e s t h e p o w - o n l y h a v e to a p p l y a c o r r e c t i o n w i t h r e s p e c t to
e r of r e s o l u t i o n . T h i s r e s u l t s in a c o m p o s i t e b u t t h e c o l o u r filter a n d t h e r e l a t i v e o p a c i t y of t h e
harmoniously merged image which cannot be o b j e c t . If a u t o m a t i o n is d e s i r e d this will h a v e to
e q u a l l e d b y a n y o t h e r a r r a n g e m e n t of i l l u m i n a - r e l a t e to t h e s e t w o factors, a n d w i t h c o n s t a n t
t i o n . F u r t h e r m o r e , t h e o p t i c a l s y s t e m is e x p o s u r e time, this can be satisfactorily
a p o c h r o m a t i c a t t h e m i r r o r focus w h i c h e n s u r e s achieved with a photosensitive glass.
e x c e l l e n t q u a l i t y of t h e i m a g e of t h e field d i a -
p h r a g m of t h e l a m p a t h i g h m a g n i f i c a t i o n s .
Systematic search for the causes of
unsatisfactory images
A D J U S T M E N T OF T H E A P E R T U R E D I A P H R A G M
A d j u s t m e n t of t h e field d i a p h r a g m is n o t p a r t i - (1) A d j u s t m e n t of t h e i l l u m i n a t i o n h a s
c u l a r l y difficult. O n c e it h a s b e e n c o r r e c t l y already been discussed.
focused in t h e o b j e c t p l a n e , o n e o n l y h a s to o p e n (2) O n e s h o u l d verify t h a t n o diffusing filter
t h e field d i a p h r a g m sufficiently to a l l o w t h e h a s b e e n p l a c e d b e t w e e n t h e field d i a p h r a g m
w h o l e field to b e i l l u m i n a t e d a n d , if n e c e s s a r y , and the condenser diaphragm.
t o a l t e r t h e p o s i t i o n of t h e c o n d e n s e r w h e n (3) O n e s h o u l d e n s u r e t h e c l e a n l i n e s s of t h e
c h a n g i n g t h e o b j e c t i v e in o r d e r to c o m p e n s a t e front s u r f a c e of t h e o b j e c t i v e b y e x a m i n i n g it
for t h e c u r v a t u r e of t h e field. T h e p o s i t i o n is w i t h a m a g n i f y i n g glass o r a n i n v e r t e d e y e p i e c e .
m o r e c o m p l e x in t h e c a s e of t h e a p e r t u r e d i a - (4) O n e s h o u l d c h e c k w h e t h e r t h e r e v o l v i n g
p h r a g m . F i r s t of all, t h e i m a g e is n o t i m - n o s e p i e c e l o c k i n g a p p a r a t u s is p o s i t i o n e d p r o p -
m e d i a t e l y a c c e s s i b l e to t h e eye b e c a u s e , in o r d e r erly.
to see it, e i t h e r t h e e y e p i e c e m u s t b e r e m o v e d (5) O n e s h o u l d verify t h a t t h e slide a n d
a n d t h e i m a g e l o o k e d for a t t h e b o t t o m of t h e c o v e r - s l i p a r e c l e a n , t h a t t h e slide is n o t u p s i d e
tube, or a n auxiliary telescope m u s t be intro- d o w n , a n d t h a t two cover-slips are not s u p e r i m -
d u c e d in p l a c e of t h e e y e p i e c e a n d a d j u s t e d a s posed.
though centring a phase diaphragm. Alterna- (6) O n e s h o u l d e n s u r e t h a t t h e w h o l e o p t i c a l
tively, if t h e m i c r o s c o p e is e q u i p p e d w i t h o n e , a s y s t e m is c l e a n , p r o c e e d i n g a s follows. L o o k i n g
B e r t r a n d lens m u s t b e i n t r o d u c e d i n t o t h e light d o w n the microscope, one rotates one eyepiece
b e a m for c o n o s c o p i c o b s e r v a t i o n . N e x t , it is a t a t i m e . If a n y d i r t is seen to r e v o l v e , it m u s t b e
n e c e s s a r y to a b o l i s h a n e x p e c t e d reflection t h a t p r e s e n t on the eyepiece a n d should therefore be
c a u s e s t h e u s e r a l m o s t i n s t i n c t i v e l y to u s e t h e c l e a n e d off. T h e n t u r n t h e b i n o c u l a r t u b e in its
a p e r t u r e d i a p h r a g m to c o n t r o l t h e light i n t e n s i - a s s e m b l y . I f m a r k s a r e seen r e v o l v i n g in r e l a t i o n
ty, w h e n it is i n a d e q u a t e . I n g e n e r a l , t h e a p e r - to the object, a n d the eyepieces are clean, the
t u r e d i a p h r a g m s h o u l d b e r e g u l a t e d in s u c h a d u s t m u s t b e in t h e b i n o c u l a r t u b e . A b i n o c u l a r
w a y t h a t b e t w e e n o n e - h a l f a n d t w o - t h i r d s of t h e t u b e m u s t never be dismantled except by an
d i a m e t e r of t h e b o t t o m lens of t h e o b j e c t i v e is expert. O n the other h a n d , the accessible entr-
i l l u m i n a t e d in t h e o b s e r v a t i o n p o s i t i o n w i t h t h e a n c e a n d exit surfaces of t h e p r i s m c a n b e
eyepiece removed. carefully c l e a n e d b y puffing a i r from a r u b b e r
W h e n v a r i a b l e focus i l l u m i n a t i o n is b r o u g h t b u l b . T h e o b j e c t i v e is t u r n e d b y slightly u n -
i n t o g e n e r a l u s e a n d v o l t a g e r e g u l a t o r s for t h e s c r e w i n g it. I f t h e e x t r a n e o u s i m a g e s also t u r n ,
30 Instruments and techniques

c h e c k t h e c l e a n l i n e s s of t h e i n t e r i o r a n d , if (19) T h e m o s t c o m m o n e r r o r in p h a s e c o n -
n e c e s s a r y , d u s t it w i t h a d r y b r u s h . t r a s t m i c r o s c o p y is failure to c e n t r e t h e i l l u m i n a -
(7) O n e s h o u l d c h e c k t h a t t h e r e is e n o u g h t i o n r i n g . I t s h o u l d b e b o r n e in m i n d t h a t it m a y
i m m e r s i o n oil, t h a t it d o e s n o t c o n t a i n b u b b l e s a s s u m e a n i n c o r r e c t p o s i t i o n b e c a u s e of t h e
o r i m p u r i t i e s a n d t h a t it d o e s n o t fluoresce. g e o m e t r y of t h e object.
(8) T h e o b j e c t i v e s h o u l d b e s c r e w e d o n cor- (20) F l u o r e s c e n c e m a y a r i s e from t h e m o u n t -
rectly. i n g m e d i u m , t h e i m m e r s i o n oil, i n a d e q u a t e o p -
(9) T h e t h i c k n e s s of t h e c o v e r - s l i p a n d of t h e tics o r from t h e w r o n g filter u s e d to c u t o u t t h e
m o u n t i n g m e d i u m s h o u l d b e c h e c k e d . If t h i s exciting radiation.
a s s e m b l y is too thick, it c a n c o m p l e t e l y p r e v e n t (21) If p o l a r i z a t i o n is u s e d a n d t h e a d j u s t -
focusing. A n i m a g e of m e d i o c r e q u a l i t y is m e n t h a s b e e n carefully c h e c k e d , s o m e d e p o l a r -
o b t a i n e d if t h e r e is too m u c h d i v e r g e n c e from i z a t i o n o r a d a r k field b e t w e e n c r o s s e d p o l a r i z e r s
t h e s t a n d a r d t h i c k n e s s (Figure 1.32). O n e s h o u l d c a n a r i s e from d u s t , a c c i d e n t a l s t r a i n s in t h e
b e c e r t a i n t h a t t h e t w o cover-slips h a v e n o t b e e n o p t i c s d u e to a b l o w o r to t h e r m a l v a r i a t i o n s , o r
used by accident. f r o m a l t e r a t i o n s in t h e p o l a r i z e r s .

76x26
22x32 22x22
18x18

F i g u r e 1.32. Standardization of microscope slides and cover-slips

(10) T h e c o n d e n s e r m o u n t i n g s h o u l d b e well- (22) A d a r k field s u c h a s a n e g a t i v e p h a s e


c e n t r e d , t h e front s h o u l d b e w e l l - s u p p o r t e d if c o n t r a s t is p a r t i c u l a r l y s e n s i t i v e to d u s t , a i r
retractable, and the ratchet should be screwed b u b b l e s in t h e m o u n t i n g o r in t h e i m m e r s i o n
d o w n sufficiently to r e m a i n in p l a c e . m e d i a , a n d to s u p e r i m p o s i t i o n of o b j e c t s in t h e
(11) T h e i l l u m i n a t i o n s h o u l d b e n e i t h e r too preparation.
w e a k n o r t o o s t r o n g , b u t o n e s h o u l d n o t a d j u s t it (23) If a m o u n t i n g m e d i u m w i t h a refractive
with the condenser d i a p h r a g m . i n d e x a l m o s t i d e n t i c a l w i t h t h a t of a c o l o u r l e s s
(12) P h a s e objectives s h o u l d n o t b e u s e d for o b j e c t is c h o s e n , t h e l a t t e r will b e i n v i s i b l e , e v e n
b r i g h t field o b s e r v a t i o n s a t h i g h p o w e r m a g n i - in p h a s e o r i n t e r f e r e n c e c o n t r a s t .
fication. (24) H a z i n e s s confined to t h e e d g e s of t h e
(13) T h e b u l b f i l a m e n t s h o u l d b e w e l l - c e n t r e d field is g e n e r a l l y d u e to insufficient c o r r e c t i o n of
in r e s p e c t of t h e c o n d e n s e r . t h e flatness of t h e field b y t h e o p t i c a l s y s t e m .
(14) T h e p l a n e side of t h e m i r r o r s h o u l d b e (25) A d a r k m a r k c r o s s i n g t h e i m a g e c a n b e
u s e d , a n d it m u s t p r o j e c t t h e b e a m o n to t h e d u e e i t h e r to a c o l l a p s e of t h e b u l b filament o r to
condenser diaphragm. a b r e a k a g e of a b u l b of t h e c o n d e n s i n g l e n s .
(15) If t h e c o n d e n s e r is r e t r a c t a b l e o r r e m o v - (26) F i e l d d a r k n e s s . T h i s p r o b l e m is c a u s e d
a b l e from t h e front, o n e s h o u l d c h e c k t h a t it h a s b y b a d c e n t r i n g of t h e i l l u m i n a t i o n . O n e s h o u l d
been positioned correctly. c h e c k t h e p o s i t i o n of t h e m i r r o r , t h e h e i g h t of t h e
(16) O n e s h o u l d u s e a c o m b i n a t i o n e y e p i e c e c o n d e n s e r a n d t h e p o s i t i o n of t h e iris d i a -
t h a t is a d a p t e d to t h e o b j e c t i v e ; in g e n e r a l , t h i s p h r a g m ; t h i s m a y b e closed d o w n t o o m u c h o r
m e a n s a v o i d i n g excessively h i g h e y e p i e c e m a g - h a v e b e c o m e n o t c e n t r e d , e s p e c i a l l y if t h e s e t - u p
nification. is s u i t a b l e for o b l i q u e i l l u m i n a t i o n . T h e r e a d e r
(17) O n e s h o u l d c o m p a r e t h e p r e p a r a t i o n s h o u l d refer to t h e r u l e s g o v e r n i n g m i c r o s c o p e
w i t h a test p r e p a r a t i o n . illumination.
(18) If t h e p o o r q u a l i t y o n l y a p p e a r s o n (27) P a r t i a l d a r k n e s s of t h e field c a n b e
photographs, there m a y have been an incorrect caused by the revolving nosepiece not being
c h o i c e of t h e o p t i c a l s y s t e m w h i c h h a s n o t b e e n t u r n e d as_far a s , o r b e i n g p u s h e d b e y o n d , t h e
c o r r e c t e d sufficiently, a n u n s a t i s f a c t o r y e m u l - s t o p n o t c h . T h e o b j e c t i v e is o n l y a t t h e c e n t r e
sion o r a w r o n g c h o i c e of c o n t r a s t filters. w h e n t h e r e s i s t a n c e of t h e s t o p n o t c h is felt.
Chief instrumental methods 31

(28) D i s p l a c e m e n t of t h e i m a g e w h e n t h e e d g e s of t h e c o v e r - s l i p . I t is p a r t i c u l a r l y a w k -
focus is v a r i e d . A p a r t from a fault in t h e m i c r o - w a r d w h e n e x a m i n i n g fresh b l o o d b e t w e e n a
s c o p e r e s u l t i n g from i n c o r r e c t c e n t r i n g o r a b n o r - slide a n d cover-slip. It can be prevented by
m a l p l a y in t h e fine a d j u s t m e n t , t h i s p r o b l e m is m a k i n g p r e p a r a t i o n s as thin as possible. T h e
generally caused by oblique illumination. T h i s less l i q u i d t h e r e is, t h e less t h e p r o b l e m c a u s e d
c a n b e r e m e d i e d b y c e n t r i n g t h e m i r r o r in o r d e r b y flow.
to p r o d u c e a x i a l i l l u m i n a t i o n , a s e x p l a i n e d p r e - B r o w n i a n or molecular m o v e m e n t con-
viously. tinuously agitates very small objects. It m u s t not
(29) P r o b l e m s of i l l u m i n a t i o n . T h e s e difficulties b e m i s t a k e n for a c t i v e m o v e m e n t , especially
m a y a r i s e from t h e a p p a r a t u s itself, s u c h a s w h e n bacteria are moving around. T h e distinc-
faulty c e n t r i n g o r d u s t o r s t a i n s o n t h e m i r r o r t i o n is n o t a l w a y s e a s y a n d n e e d s m i n u t e a t t e n -
a n d c o n d e n s e r l e n s e s . T h e y m a y b e d u e to tion.
external objects such as w i n d o w b a r s or objects
a c c i d e n t a l l y i n t e r p o s e d in t h e p a t h of t h e b e a m
of light. I n b o t h c a s e s t h e n a t u r e of t h e p r o b l e m
c a n b e d e t e r m i n e d b y slightly d i s p l a c i n g t h e 1.3 Chief instrumental
c o n d e n s e r b y m e a n s of t h e r a t c h e t : t h e i l l u m i n a - methods
t i o n is i m m e d i a t e l y s e e n to i m p r o v e a n d t h e
s o u r c e of t h e p r o b l e m c a n b e d i s c o v e r e d .
Dark ground illumination
W h e n t h e c o n e of l i g h t s u p p l i e d b y t h e c o n -
d e n s e r is t o o l a r g e , i.e. w h e n t h e iris d i a p h r a g m V a r i o u s m i c r o s c o p i c m e t h o d s a r e u s e d for
is o p e n t o o w i d e , t h e d e t a i l s of t h e o b j e c t , o b s e r v i n g a n o b j e c t o n a d a r k field, t h a t is to s a y
e s p e c i a l l y if it is c o l o u r l e s s , a r e s w a m p e d b y u t i l i z i n g l i g h t diffracted from it: (a) i n c i d e n t o r
excessive b r i g h t n e s s a n d t h e y m a y b e c o m e in- reflected l i g h t w i t h o b l i q u e o r a n n u l a r light; (b)
distinct or disappear altogether. This phe- e x a m i n a t i o n b y t r a n s m i t t e d light, u s i n g a c e n -
n o m e n o n o c c u r s b e c a u s e t h e light b e a m r e a c h e s t r a l o r a n n u l a r s c r e e n in t h e o b j e c t i v e to s t o p
t h e o b s e r v e r ' s eye d i r e c t l y , w i t h o u t t r a v e r s i n g d i r e c t light of z e r o o r d e r ( c e n t r a l d a r k field); (c)
t h e o b j e c t , a s a r e s u l t of reflections o n t h e slide, e x a m i n a t i o n b y t r a n s m i t t e d light, u s i n g a n n u l a r
c o v e r - s l i p , a i r b u b b l e s in t h e p r e p a r a t i o n , o b j e c - l i g h t from a n u m e r i c a l a p e r t u r e g r e a t e r t h a n
tive m o u n t i n g , t u b e w a l l s , e t c . T h e s e reflections t h a t of t h e lens ( a n n u l a r d a r k field); (d) ex-
c h a n g e t h e c l a r i t y of t h e i m a g e to a g r e a t e r o r a m i n a t i o n of a b i r f r i n g e n t o b j e c t b e t w e e n t w o
lesser e x t e n t . T h e y c a n b e easily r e m e d i e d b y c r o s s e d p o l a r i z e r s ; (e) e x a m i n a t i o n a t r i g h t
c o r r e c t i l l u m i n a t i o n a n d b y c l o s i n g first t h e field a n g l e s to t h e d i r e c t i o n of t h e b e a m of light in a
diaphragm and then the aperture diaphragm flat field ( u l t r a m i c r o s c o p i c d a r k field).
until the i m a g e becomes clear. E x a m i n a t i o n b y reflected light w i t h o b l i q u e o r
(30) S u b j e c t i v e p r o b l e m s T h e s e difficulties m a y a n n u l a r i l l u m i n a t i o n is m o s t c o m m o n l y u s e d
a p p e a r a s i m a g e s of r o u n d c o l o u r l e s s b o d i e s , with stereoscopic microscopes or binocular
s h a p e d like a s t r i n g of b e a d s o r in t h e f o r m of m a g n i f i e r s . A s a r u l e , w h e n t h e o b j e c t fills t h e
filaments, w h i c h a p p e a r to m o v e a b o u t in t h e w h o l e of t h e field of o b s e r v a t i o n , t h e d a r k field is
m i c r o s c o p i c field. W i t h a little p r a c t i c e , b y not perceived.
slightly m o v i n g t h e p r e p a r a t i o n o r b y a l t e r i n g E x a m i n a t i o n b y t r a n s m i t t e d light, u s i n g a
t h e focus, it c a n b e seen t h a t t h e s e f o r m a t i o n s central screen, requires a n objective with a n
a r e i n d e p e n d e n t of t h e o b j e c t e x a m i n e d a n d of internal Spierer screen. W i t h an a n n u l a r screen,
t h e m i c r o s c o p e . G e n e r a l l y t h e y c o n s i s t of cellu- t h a t of W i l s k a s h o u l d b e u s e d . A n o p t e r a l , t h e
l a r d e b r i s w h i c h float in t h e a q u e o u s h u m o u r o r i g i n a l v e r s i o n , is p r e f e r r e d to t h e i n d u s t r i a l
a n d p r o j e c t o n to t h e r e t i n a . T h e i m a g e s to instrument which has a negative phase contrast
w h i c h t h e y give rise o r i g i n a t e in t h e o b s e r v e r ' s with intense absorption.
eye. S o m e t i m e s t h e y a r e v e r y t r o u b l e s o m e , a n d E x a m i n a t i o n b y t r a n s m i t t e d light w i t h a n n u -
c a n o n l y b e e l i m i n a t e d , a t least t e m p o r a r i l y , b y l a r i l l u m i n a t i o n from a n a p e r t u r e g r e a t e r t h a n
rest. t h a t of t h e o b j e c t i v e is t h e o n l y m e t h o d n o r m a l l y
(31) F l o w a n d B r o w n i a n m o v e m e n t . F l o w o c c u r s r e f e r r e d to a s d a r k g r o u n d . I t s h o u l d b e n o t e d
p a r t i c u l a r l y in u n m o u n t e d p r e p a r a t i o n s ; it is t h a t it is v e r y difficult to u s e p o w e r f u l objectives
c a u s e d e i t h e r b y t h e m i c r o s c o p e b e i n g tilted, o r h a v i n g a n u m e r i c a l a p e r t u r e g r e a t e r t h a n 1.0 o n
b y d r y i n g u p , o r b y t h e a d d i t i o n of l i q u i d s a t t h e a d a r k field, s i n c e t h e c o n d e n s e r m u s t a l w a y s
32 Instruments and techniques

Figure 1.33. Diffraction on a dark field. The


direct beam no longer enters the objective and
only the diffracted beams participate in image
formation, because of the oblique illumination

h a v e a n a p e r t u r e greater t h a n this value. It m u s t


be immersed without e n t r a p p i n g any air b u b -
b l e s , w h i c h is difficult to d o (Figures 1.33 a n d
1.34).
E x a m i n a t i o n of a b i r f r i n g e n t o b j e c t b e t w e e n
c r o s s e d p o l a r i z e r s o n a d a r k field gives a n i m a g e
of a r a t h e r s p e c i a l n a t u r e (see l a t e r in t h i s
s e c t i o n ) . E x a m i n a t i o n u s i n g a light b e a m p e r -
p e n d i c u l a r to t h e d i r e c t i o n of o b s e r v a t i o n ,
F i g u r e 1.35. Images on a bright field and a dark field of
a c c o r d i n g to Z i e d e n t o p f a n d S z i g m o n d y , r e s u l t s
different diatoms (Leitz-Wetzlar)
in t h e o c c u r r e n c e of t h e ' u n i a z i m u t h a l effect'
a n d hence provides information about the n u m e r o u s diffraction fringes, is p r o d u c e d if t h e
o r i e n t a t i o n of t h e p a r t i c l e s o b s e r v e d . o b j e c t refracts l i g h t . T h e full s p a c e s in t h e c e n t r e
of a s t r u c t u r e a p p e a r o p t i c a l l y e m p t y . T h e r e -
N A T U R E A N D S T R U C T U R E OF IMAGES F O R M E D fore, c a r e m u s t b e t a k e n in i n t e r p r e t i n g t h e
O N A D A R K FIELD i m a g e s . S i n c e t h e i m a g e is o n l y f o r m e d b y
I m a g e s f o r m e d o n a r e a l d a r k field a r e r e m a r k - diffracted light, w h i c h is a l w a y s w e a k e r t h a n
a b l e in t h a t a n e d g e effect, w i t h v e r y b r i g h t a n d d i r e c t light, v e r y i n t e n s e s o u r c e s m u s t b e u s e d

F i g u r e 1.34. T w o types of
condenser on a dark field.
Leftimmersion of the front
surface; rightwithout
immersion
Chief instrumental methods 33

a n d t h i s is n o t a l w a y s c o n v e n i e n t if t h e s p e c i - first m a d e of p o l i s h e d silver t h e n of silvered


m e n s a r e alive a n d , c o n s e q u e n t l y , v e r y s e n s i t i v e g l a s s , t h e focus of w h i c h w a s t h e s a m e a s t h a t of
t o p o w e r f u l light (Figure 1.35). t h e lens for w h i c h it w a s m a d e a n d w h i c h w a s
m o u n t e d o n its f r a m e . T h e r e f o r e , a n e q u a l n u m -
b e r of m i r r o r s a n d lenses w a s u s e d . T h i s a p p a r -
Examination u n d e r reflected light
a t u s , i l l u m i n a t e d from b e l o w b y light reflected
M i c r o s c o p i s t s h a v e a l w a y s a t t e m p t e d to ex- b y t h e m i r r o r of t h e m i c r o s c o p e , g a v e excellent
a m i n e o p a q u e objects, such as crystals or small r e s u l t s a n d it is h a r d to see w h y it h a s b e e n
organisms, b u t such n o n - t r a n s p a r e n t objects f o r g o t t e n . P e r h a p s it a r i s e s from t h e fact t h a t
h a v e to b e i l l u m i n a t e d b y reflected light. W i t h sections a n d t r a n s p a r e n t p r e p a r a t i o n s are easier
l o w p o w e r m a g n i f i c a t i o n , a s u i t a b l y p l a c e d lens t o o b s e r v e a n d i n t e r p r e t , a n d b e c a u s e of t h e
w a s sufficient, b u t w h e n u s i n g h i g h p o w e r a l m o s t e x c l u s i v e u s e of t r a n s m i t t e d light in
lenses, a n o t h e r appliance was required. As early microscopy.
a s 1668, L e e u w e n h o e k e q u i p p e d his lenses w i t h Lieberkiihn's m i r r o r h a s , therefore, been
a c o n c a v e reflector m a d e of p o l i s h e d c o p p e r . I n s u p e r s e d e d b y i n c i d e n t i l l u m i n a t o r s , also k n o w n
1740, L i e b e r k h n i n v e n t e d t h e m i r r o r w h i c h a s o p a q u e i l l u m i n a t o r s , w h i c h e n a b l e t h e ex-
n o w b e a r s his n a m e . I t w a s a c o n c a v e m i r r o r , a m i n a t i o n of o p a q u e o b j e c t s u n d e r reflected

Figure 1.36. Illumination of an opaque object through the objective on the left, around the objective on the right using the same
objective with an overhead angular condenser (Wild-Heerbrugg)
34 Instruments and techniques

light, e v e n w i t h p o w e r f u l s h o r t - f o c u s l e n s e s . I n T h e o b j e c t i v e s a r e c o r r e c t e d for a t u b e l e n g t h
t h e s e i n s t r u m e n t s , i l l u m i n a t i o n is p r o d u c e d of 185 m m to c o m p e n s a t e for t h e e x t e n s i o n
t h r o u g h t h e o b j e c t i v e b y m e a n s of a t o t a l l y p r o d u c e d in t h e n o r m a l 170 m m t u b e a s a r e s u l t
reflecting p r i s m o r of a semi-reflecting m i r r o r of t h e l e n g t h of t h e U l t r o p a k i n s t r u m e n t . T h e y
p l a c e d a b o v e t h e objectives a n d i l l u m i n a t e d h a v e a l o n g w o r k i n g d i s t a n c e w h i c h m a k e s it
from t h e side b y a n a p p r o p r i a t e d e v i c e . T h e s e p o s s i b l e to o b s e r v e o b j e c t s in d e p t h , l a y e r b y
i n s t r u m e n t s a r e w i d e l y u s e d in m e t a l l u r g y ; l a y e r . T h e front lenses a r e o b l o n g , b u t t h e i r
o t h e r w i s e t h e y a r e only s u i t a b l e for s t u d y i n g edges are protected against penetration by rays
p o l i s h e d s u r f a c e s , b e c a u s e i r r e g u l a r surfaces of light n o t p a r t i c i p a t i n g in i m a g e f o r m a t i o n .
p r o d u c e reflections. T h i s e x p l a i n s w h y t h e o p a - T h e a p e r t u r e of t h e front lens c o r r e s p o n d s e x a c t -
q u e i l l u m i n a t o r is s e l d o m u s e d in biology (Figure ly to t h e n u m e r i c a l a p e r t u r e of t h e o b j e c t i v e a n d
1.36). t o t h e size of t h e o p t i c a l field.
U s u a l l y , t h e s e objectives m u s t b e u s e d w i t h -
ULTROPAK o u t cover-slips, especially the high power objec-
I n b i o l o g y , e x a m i n a t i o n b y reflected light h a s tive, X 5 0 ; h o w e v e r , a c o v e r - s l i p m a y b e u s e d
r e g a i n e d its w e l l - d e s e r v e d p l a c e a m o n g m e t h o d s with low magnification with w a t e r i m m e r s i o n
for e x a m i n i n g living tissues. L e i t z h a v e r e v i v e d a n d , in p a r t i c u l a r , w i t h h o m o g e n e o u s i m m e r -
a n d i m p r o v e d t h e c o n c e p t i o n of L i e b e r k h n ' s s i o n . T h e s e last t y p e s of o b j e c t i v e c a n b e u s e d in
m i r r o r , i.e. e p i - i l l u m i n a t i o n , n o t a c r o s s t h e lens b o t h t r a n s m i t t e d light a n d in reflected light.
as with the o p a q u e illuminator. In these instru- O b j e c t i v e s of l o w p o w e r h a v e a n a n n u l a r c o n -
m e n t s , p e r i p h e r a l a n n u l a r i l l u m i n a t i o n is p r o - denser, whereas those with higher power have a
d u c e d b y a n a n n u l a r c o n d e n s e r w i t h objectives m i r r o r c o n d e n s e r . T h e s e different c o n d e n s e r s
o r w i t h a m i r r o r c o n d e n s e r (Figure 1.36). c a n b e a d j u s t e d w i t h a s p e c i a l s c r e w ; t h e illu-
L e i t z d e s i g n e d a series of 15 objectives w i t h m i n a t i o n c a n t h u s b e a d j u s t e d a c c o r d i n g to t h e
i n t r i n s i c m a g n i f i c a t i o n s , v a r y i n g from 3 , 8 to n a t u r e of t h e o b j e c t s , t h e m e a n p o s i t i o n b e i n g
100, e a c h e q u i p p e d w i t h a n a n n u l a r c o n d e n s e r s h o w n b y a r e d m a r k . T h e objectives c a n b e
with an objective or a mirror having the s a m e s t o p p e d d o w n to i n c r e a s e t h e p e n e t r a t i n g p o w e r .
focal l e n g t h as t h e o b j e c t i v e . Six c a n b e u s e d d r y T h e r e is a series of 6 d i a p h r a g m s w i t h d i a m e t e r s
( U - O 3.8, 5, 6.5, 1 1 , 22, 5 0 ) , 4 for w a t e r i m m e r - of 5, 4, 3 , 2 . 5 , 2 a n d 1.5 m m , r e s p e c t i v e l y . T h e
sion ( U - O 2 3 , 5 5 , 70, 90) a n d 5 for oil i m m e r s i o n c h o i c e of d i a p h r a g m d e p e n d s o n t h e n a t u r e of
( U - O 1 1 , 2 3 , 6 0 , 75, 100). I l l u m i n a t i o n is p r o - t h e o b j e c t . A n excessively n a r r o w d i a p h r a g m
d u c e d l a t e r a l l y b y m e a n s of a s m a l l , low v o l t a g e interferes with resolution; they are used with or
i n c a n d e s c e n t b u l b , t h e light of w h i c h is d i r e c t e d w i t h o u t a s p e c i a l t u b e ; for e x a m p l e , n o s . X 3 . 8
b y a s m a l l c o n d e n s e r o n t h e reflecting surfaces a n d X 11 for h o m o g e n e o u s i m m e r s i o n a r e u s e d
w h i c h i l l u m i n a t e t h e objectives o r a n n u l a r m i r - w i t h o u t a tube; no. X 1 I T (with a dry objective),
rors. X 2 2 w i t h t u b e 2 2 a n d so o n . A s e c t o r
d i a p h r a g m w h i c h p a s s e s from 90 to 180 m a k e s
it p o s s i b l e to o b t a i n c e r t a i n i l l u m i n a t i o n effects;
it is i n s e r t e d i n t o t h e h o l d e r for t h e filters a n d
screens.

I M M E R S I O N O R D I P P I N G APPLIANCES
T h e s e a p p l i a n c e s a r e u s e d for o b s e r v a t i o n of
m o i s t o b j e c t s , m u c u s o r fat ( o r g a n s , fish, e t c . ) ,
w h i c h h a v e i r r e g u l a r surfaces g i v i n g rise to
reflections w h i c h interfere w i t h o b s e r v a t i o n s .
T h e u s e of a c o v e r - s l i p is insufficient, e s p e c i a l l y
w i t h low m a g n i f i c a t i o n , b e c a u s e of t h e reflec-
tions p r o d u c e d by the cover-slip or by the
surface irregularities. D i p p i n g devices are conic-
al in s h a p e a n d e l o n g a t e d to s o m e e x t e n t . T h e i r
Figure 1.37. Zetopan research microscope. The light path
f r o n t a l surface m a y b e in c o n t a c t w i t h t h e l i q u i d
under transmitted light and with annular incident light. A,
plane of aperture diaphragm (Reichert, Vienna); B, plane of a t t h e surface of t h e object. T h e i r u p p e r s u r f a c e
field diaphragm is l o c a t e d sufficiently h i g h to p r e v e n t s t r a y l i g h t
Chief instrumental methods 35

from p e n e t r a t i n g . T h e s e a p p l i a n c e s a r e i n d e p e n - T h e s u r f a c e of t h e s e c t i o n is s t a i n e d w i t h a 1 %
d e n t of t h e a n n u l a r i l l u m i n a t i o n a n d c a n b e s o l u t i o n of t o l u i d i n e b l u e in m e t h a n o l , a p p l i e d
a d j u s t e d a c c o r d i n g to t h e n a t u r e of t h e o b j e c t , w i t h a b r u s h . T h e s o l u t i o n is left for 5 0 - 6 0 s in
w h i c h t h e y c a n flatten. T h e y c a n a l s o b e d i p p e d t h e c a s e of a fresh s p e c i m e n , a n d a few s e c o n d s
to varying d e p t h s into water or other liquids. w h e n d e a l i n g w i t h a p r e p a r a t i o n fixed w i t h
T h e y a r e a v a i l a b l e for n o s . X 3 . 8 , X 5 , X 6 . 5 a n d f o r m a l i n . O n l y t h e superficial cell l a y e r n e e d s to
X11 objectives. T h e s e objectives can be used b e s t a i n e d . T h e c o v e r of t h e d i s h , w h i c h h a s a n
dry, immersed or dipped. i n s p e c t i o n g l a s s w i n d o w , is t h e n a d j u s t e d w i t h
W a t e r o r oil i m m e r s i o n o b j e c t i v e s a r e i n - t h e r i n g . T h e r i n g m a k e s it p o s s i b l e to p r e s s t h e
t e n d e d for d i r e c t o b s e r v a t i o n of o b j e c t s w i t h o r g l a s s of t h e w i n d o w a g a i n s t t h e surface of t h e
w i t h o u t c o v e r - s l i p s . T h e l a t t e r is u s e d to flatten s e c t i o n to e x p e l excess s t a i n . I f n e c e s s a r y , a d r o p
o u t t h e o b j e c t o r to p r o t e c t t h e s p e c i m e n o r t h e of g l y c e r i n c a n b e p l a c e d o n t h e w i n d o w to
o b j e c t i v e . S p e c i a l c a p s w i t h c o v e r - s l i p s of 0.15 e n s u r e c o n t a c t w i t h t h e U l t r o p a k objectives.
- 0 . 3 m m t h i c k n e s s a r e a v a i l a b l e for t h e s e o b j e c - T h e m o s t useful m a g n i f i c a t i o n s g i v e n b y U l t r o -
tives. T h e y m u s t b e m a d e to a d h e r e to t h e p a k o b j e c t i v e s a r e X 6 a n d X l l in c o m b i n a t i o n
o b j e c t i v e b y a d r o p of i m m e r s i o n l i q u i d . T h e s e w i t h a X 8 e y e p i e c e . F o r v e r y low m a g n i f i c a -
c a p s m a y a l s o b e u s e d o n d r y o b j e c t i v e s , for tions, objectives X 1 a n d X 4 are used with a X6
e x a m p l e , w i t h infectious m a t e r i a l . e y e p i e c e . T h e m e t a c h r o m a s y of t o l u i d i n e b l u e
I t is n e c e s s a r y to u s e slides m a d e of b l a c k p r o d u c e s beautiful staining with varied shades.
g l a s s w h i c h c u t o u t l a t e r a l reflections o r reflec- I n t h e c a s e of fixed s p e c i m e n s , o n e n e e d o n l y
t i o n s from u n d e r n e a t h for o b j e c t s s m a l l e r t h a n s t a i n for a few s e c o n d s . S p e c i m e n s fixed w i t h
t h e o p t i c a l field. formalin can be used directly. Specimens which
Excellent results m a y be obtained, as we h a v e h a v e b e e n fixed w i t h B o u i n ' s fixative m u s t first
b e e n a b l e to c o n f i r m . S i n c e t h e r a y s of l i g h t n o b e b l e a c h e d w i t h a c o n c e n t r a t e d a q u e o u s solu-
longer pass t h r o u g h the objective, b u t outside t i o n of s o d i u m b e n z o a t e for 12 h in a d r y i n g o v e n
a n d a r o u n d it, t h e r e is n o l o n g e r a n y d a n g e r of at 56 C.
s t r a y reflections w h i c h c a u s e s u c h p r o b l e m s A f t e r s t a i n i n g , b o t h fresh a n d fixed p r e p a r a -
w h e n u s i n g t h e o p a q u e i l l u m i n a t o r , a n d t h e full t i o n s m a y b e w a s h e d v e r y carefully w i t h distilled
a p e r t u r e of t h e o b j e c t i v e s m a y b e u s e d to s t u d y w a t e r u s i n g a b r u s h , a n d t h e n differentiated
the specimen. with dilute picric acid.
W e a r e c o n v i n c e d t h a t t h e p r a c t i c a l u s e of t h i s
m e t h o d of i l l u m i n a t i o n c o n s t i t u t e s a s g r e a t a LEROUX-CHAMPEAU METHOD
p r o g r e s s a s i m p r o v e m e n t s in d a r k g r o u n d illu- T h e n e w t e c h n i q u e of d i r e c t e x a m i n a t i o n of
m i n a t i o n . I n t h i s w a y , it will b e p o s s i b l e to see, s p e c i m e n s e m b e d d e d in paraffin, w i t h U l t r o p a k ,
u n d e r a m i c r o s c o p e , t h e t r u e a p p e a r a n c e of is a v e r y i n t e r e s t i n g a p p l i c a t i o n of t h e L e r o u x
m a n y objects a n d tissue specimens w i t h o u t hav- m e t h o d . F i r s t , o n e s i d e of t h e block of paraffin
i n g to flatten t h e m o u t o r to slice t h e m i n t o t h i n m u s t b e p o l i s h e d u n t i l its surface is perfectly
s e c t i o n s to m a k e t h e m t r a n s p a r e n t . I t will b e s m o o t h a n d s h i n y , u s i n g a p o l i s h i n g s t o n e of
p o s s i b l e t o s t u d y living tissues w i t h o u t a n y semi-fine t e x t u r e , w e t t e d w i t h 9 5 % a n h y d r o u s
c h a n g e s . I t is a s s i m p l e to p h o t o g r a p h t h e s e e t h a n o l . T h e s u r f a c e is a b r a d e d w h i l e r o t a t i n g .
images as o r d i n a r y microscopical p r e p a r a t i o n s . I t is r i n s e d u n d e r t h e t a p , a n d p o l i s h i n g is
c o n t i n u e d w i t h a v e r y fine w h e t s t o n e , w e t t e d
w i t h e t h a n o l . T h e p o l i s h i n g is c o m p l e t e d o n a
M E T H O D OF R. L E R O U X
r o t a t i n g g l a s s s l a b o r a s h e e t s o a k e d in e t h a n o l .
I n 1 9 3 1 , R . L e r o u x d e v i s e d a n e x t r e m e l y in-
O f c o u r s e , t h e surface of t h e s p e c i m e n m u s t b e
teresting s p o n t a n e o u s histological e x a m i n a t i o n
flush so t h a t it c a n b e s t a i n e d . T h e block is t h e n
u s i n g U l t r o p a k . T h e m a t e r i a l to b e e x a m i n e d ,
fixed to t h e b o t t o m of t h e m a c r o t o m e d i s h w i t h a
fresh o r fixed, is p a r t i a l l y solidified b y s p r a y i n g
few d r o p s of w a t e r . T h e c o v e r w i t h t h e w i n d o w
it w i t h l i q u i d c a r b o n d i o x i d e a t t h e b o t t o m of a
is of n o u s e in t h i s c a s e . T h e surface is s t a i n e d
special dish k n o w n as a m a c r o t o m e dish, m a d e
with a b r u s h as above, a n d the e x a m i n a t i o n with
b y L e i t z . T h e surface of t h e s p e c i m e n is t h e n
U l t r o p a k can be carried on with or without a
flattened o u t w i t h a l a r g e m i c r o t o m e knife. T h e
c o v e r - s l i p a n d a c o n t a c t l i q u i d . O n l y t h e surface
knife is g u i d e d b y a r i n g t h e h e i g h t of w h i c h c a n
l a y e r of cells is s t a i n e d a s in t h e c a s e of n o n -
b e a d j u s t e d w i t h a s c r e w l o c a t e d a t t h e t o p of t h e
embedded specimens.
cell.
36 Instruments and techniques

J . L . Luntley has proposed a very simple in t h e l a m p to o b t a i n a g o o d o u t p u t of i n f r a r e d


p r o c e s s u n d e r reflected light. I t involves t h e u s e light.
of a s m a l l p o c k e t t o r c h b u l b of 2.5 V o r 3.5 V S p e c i a l t r a n s p o s i t i o n c o l o u r films, a s u s e d in
a t t a c h e d to t h e o b j e c t - s t a g e b y a b r a s s s t a p l e a e r i a l p h o t o g r a p h y , e n a b l e s o n e to o b t a i n v e r y
w i t h a s u i t a b l e s m a l l s l i d i n g sleeve. A b u l b instructive photomicrographs covering both the
p r e f e r a b l y w i t h thick glass a t t h e front is c h o s e n , visible a n d i n f r a r e d r e g i o n s o n w h i c h a r b i t r a r y
a s it a c t s a s a c o n d e n s e r . P o w e r is s u p p l i e d b y a c o l o u r of t h e visible r e g i o n t r a n s l a t e s t h e in-
small powerful torch battery or by a small frared.
transformer. I n t h e a b s e n c e of a n i m a g e c o n v e r t e r for
f o c u s i n g , o n e c a n t a k e a series of p i c t u r e s a t
Infrared microscopy 1 i n t e r v a l s , m e a s u r e d o n t h e scale g r a d u a -
t i o n s u s i n g t h e fine a d j u s t m e n t . After d e v e l o p -
I n c r e a s i n g t h e w a v e l e n g t h of light d e c r e a s e s t h e
i n g , a p e r m a n e n t n o t e is m a d e of t h e c o r r e c t
r e s o l v i n g p o w e r of t h e o p t i c a l s y s t e m , so t h a t
focus a n d of t h e c o r r e s p o n d i n g d i s p l a c e m e n t .
o b s e r v a t i o n s u n d e r i n f r a r e d light d o n o t f a v o u r
H o w e v e r , o n e h a s to r e p e a t t h e o p e r a t i o n for
h i g h m a g n i f i c a t i o n s . S o m e o b j e c t s , especially if
e v e r y o b j e c t i v e , especially if t h e m a g n i f i c a t i o n is
strongly impregnated with mlanine, are highly
different.
o p a q u e to visible light b u t fairly t r a n s p a r e n t
u n d e r i n f r a r e d light, e.g. c o l e o p t e r a i n t e g u m e n t s
Ultraviolet microscopy
o r fossilized s t o m a c h s of c h i t i n o z o a . I n s u c h
c a s e s e x a m i n a t i o n u n d e r i n f r a r e d l i g h t is T h e r e a r e t w o m a j o r r e a s o n s in f a v o u r of u s i n g
o b v i o u s l y of i n t e r e s t . S i n c e o b j e c t i v e s , e x c e p t for u l t r a v i o l e t i l l u m i n a t i o n for m i c r o s c o p e s : t h e r e -
those comprising mirrors, cannot be corrected s o l v i n g p o w e r c a n b e d o u b l e d , a n d selective
for i n f r a r e d light, o n e m u s t focus u n d e r i n f r a r e d a b s o r p t i o n s o c c u r a t v e r y specific w a v e l e n g t h s
l i g h t before r e c o r d i n g t h e i m a g e o n a n e m u l s i o n b y different cell c o n s t i t u e n t s , s u c h a s r i b o n u c l e i c
s e n s i t i v e to i n f r a r e d . T h i s is p e r f o r m e d u s i n g a c i d s . I n p r a c t i c e , m a n y o b s t a c l e s h a v e to b e
image converter tubes. T h e s e tubes are inter- o v e r c o m e before t h e s e m e t h o d s receive t h e r e c -
c h a n g e a b l e w i t h u l t r a v i o l e t c o n v e r t e r s in m o d - o g n i t i o n t h e y d e s e r v e . F i r s t , s o u r c e s a r e costly,
e r n e q u i p m e n t , w h i c h m a k e s it p o s s i b l e to focus often v e r y u n s t a b l e a n d d a n g e r o u s to t h e eye of
o u t s i d e t h e visible r a n g e . A s far a s i l l u m i n a t i o n t h e o p e r a t o r . T h e o b j e c t i v e s m u s t b e specially
is c o n c e r n e d , t h e i n f r a r e d r e g i o n is selected w i t h corrected; they usually have a mirror or mixed
g l a s s filters o r i n t e r f e r e n c e filters, if a n a r r o w reflecting ( c a t a d i o p t r i c ) o b j e c t i v e s ; t h e i r reflect-
b a n d is r e q u i r e d . I t is i m p o r t a n t to r e m o v e all i n g l a y e r s a g e v e r y r a p i d l y d u e to t h e c a t a l y t i c
h e a t - a b s o r b i n g filters o r m i r r o r s from t h e i l l u m i - effect of t h e u l t r a v i o l e t r a d i a t i o n . T h i s c a u s e s
n a t i n g s y s t e m r a t h e r t h a n u s e too little c u r r e n t t h e reflecting p o w e r to d e c r e a s e r a p i d l y a n d t h e

I m a g e plane

Object plane

Figure 1.38. Four types of reflecting objective for the visible ultraviolet and infrared ranges based on the Scharzschild
system. Left to rightobjectives with increasing magnification
Chief instrumental methods 37

o b j e c t i v e to b e c o m e u n u s a b l e . I n s e v e r a l c a s e s , D I F F E R E N T I A L D I S P E R S I O N BY D I F F R A C T I O N
s o m e of t h e o b j e c t i v e lenses m a y e v e n c h a n g e a s T H E BERECK EFFECT
a r e s u l t of t h e h a r m f u l a c t i o n of u l t r a v i o l e t W h e n a filamentous s t r u c t u r e like a flagellum o r
r a d i a t i o n (Figure 1.38). a c i l i u m is i m m e r s e d in a m e d i u m of different
T h e most frequently used sources are high r e f r a c t i v e i n d e x , its e d g e s s h o w a differential
pressure mercury vapour quartz lamps, hyd- a p p a r e n t colour, which varies with the m e d i u m
rogen lamps, and arc lamps with rotating cad- a n d its t e m p e r a t u r e . T h i s effect, w h i c h w a s
m i u m e l e c t r o d e s u s e d in c o n j u n c t i o n w i t h n a r - o b s e r v e d first b y B e r e c k , is e x a g g e r a t e d b y t h e
r o w b a n d filters or, p r e f e r a b l y , w i t h a n u l t r a - r e s i d u a l c h r o m a t i c a b e r r a t i o n of s o m e o b j e c -
violet m o n o c h r o m a t o r . tives. P a r a d o x i c a l l y , it is e a s i e r to o b s e r v e w i t h
M i r r o r o b j e c t i v e s ( p u r e reflectors) h a v e a an achromatic than with an apochromatic
g r e a t a d v a n t a g e o v e r o t h e r t y p e s in t h a t o n e c a n objective.
focus t h e m in visible light a n d t h e i r focus r e - T h e B e r e c k effect is well seen in cell g h o s t s ,
m a i n s u n c h a n g e d u n d e r ultraviolet light, w h o s e c o n t e n t s h a v e b e e n lysed o u t l e a v i n g t h e i r
i r r e s p e c t i v e of t h e w a v e l e n g t h . F o r o t h e r o b j e c - capsules. T h e i r weak birefringence m a k e t h e m
tives, a c o r r e c t i o n m u s t g e n e r a l l y b e m a d e w i t h difficult to see. A s o n e c h a n g e s t h e refractive
r e s p e c t to t h e g r e e n m e r c u r y line u s i n g a fine i n d e x of t h e i m m e r s i o n m e d i u m , t h e y b e c o m e
a d j u s t m e n t w i t h a n a c c u r a c y of less t h a n 0.1 . i n v i s i b l e in m o n o c h r o m a t i c light a t a v e r y p r e -
F o r this reason, s o m e s t a n d s especially designed cise refractive i n d e x . I n w h i t e light, t h i s v a l u e of
for u l t r a v i o l e t m i c r o s c o p y h a v e t h r e e s e t t i n g s t h e i n d e x gives a m a x i m u m B e r e c k effect. T h e
c o a r s e , fine a n d u l t r a - f i n e focusing. list b e l o w gives p a i r s of fluids r e c o m m e n d e d b y
Table 1.4 in S e c t i o n 1.12 s h o w s t h e m o s t F r e y - W y s s l i n g to b e m i x e d to give m e d i a of
commonly used ultraviolet b a n d s and their a p - p a r t i c u l a r refractive i n d i c e s :
p l i c a t i o n s . N e a r l y all p r o t e i n s a r e o p a q u e b e l o w W a t e r 1.333 p l u s g l y c e r i n 1.461
0.250 , so t h a t t h e 0 . 2 7 5 - 0 . 3 7 5 w i n d o w is G l y c e r i n 1.461 p l u s q u i n o l i n e 1.613
m o s t useful. O n l y g l y c e r i n is t r a n s p a r e n t in t h e A c e t o n e 1.362 p l u s b e n z y l i o d i d e 1.624
w h o l e of t h e u l t r a v i o l e t r a n g e e m p l o y e d . I t m u s t B e n z y l i o d i d e 1.624 p l u s m e t h y l e n e i o d i d e 1.739
b e u s e d exclusively a s m o u n t i n g o r i m m e r s i o n W a t e r 1.333 p l u s m e r c u r y p o t a s s i u m i o d i d e
m e d i u m ; c o v e r - s l i p s a n d slides m u s t b e m a d e of 1.721
q u a r t z . T h e r e s o l v i n g p o w e r is g i v e n in Figure
1.13. A c e t o n e 1.362 p l u s m e t h y l e n e i o d i d e 1.739

T E C H N I Q U E S O F C A R R I C A B U R U (1967) U S I N G Pupillary modulators


M O N O C H R O M A T I C POLARIZED INFRARED T h e f u n d a m e n t a l e x p e r i m e n t s of A b b e s h o w e d
LIGHT t h a t t h e r e a r e t w o p r e f e r r e d p l a n e s in t h e o b j e c -
T h e s e t e c h n i q u e s c a n b e e m p l o y e d to s t u d y tives of a m i c r o s c o p e t h e front focal p l a n e in
m e l a n i n s p e c i m e n s , w h i c h a r e o p a q u e t o visible w h i c h t h e o b j e c t is l o c a t e d w h e n t h e o b j e c t i v e
l i g h t . O n e s h o u l d b e a r in m i n d t h a t n o r m a l p r o j e c t s its i m a g e i n t o infinity, a n d t h e r e a r focal
p o l a r i z i n g filters c a n n o t b e u s e d for i n f r a r e d p l a n e o r i n t e r n a l focal p l a n e w h e r e t h e diffrac-
l i g h t ; s p e c i a l filters a r e r e q u i r e d . T h e i m a g e s a r e t i o n i m a g e of t h e o b j e c t o r h o l o g r a m is f o r m e d
s e e n b y t r a n s f o r m i n g t h e light w h i c h affects b y F o u r i e r t r a n s f o r m a t i o n . T h e l a t t e r is
p l a t e s s e n s i t i v e to i n f r a r e d . I n s e c t s w i t h m e l a n i n o b s e r v e d in c o n o s c o p y a n d t h e f o r m e r r e c o r d e d
a n d D e m a t i a fungi c a n b e m a d e t r a n s p a r e n t , in o r t h o s c o p y . W h e n s t u d y i n g t h e d i s t r i b u t i o n
a n d their anisotropy examined without chemical of a m p l i t u d e s a n d p h a s e s in t h e r e a r focal p l a n e
d e c o l o r a t i o n , b u t o n e s h o u l d a l w a y s b e a w a r e of of a p o w e r f u l o b j e c t i v e , a n e s s e n t i a l l y m a r g i n a l
c h a n g e s in t h e m . k i n d of m o d u l a t i o n m a y b e d e t e c t e d . T h i s m o d -
u l a t i o n , d u e to t h e s t r u c t u r e of t h e o b j e c t i v e , c a n
b e u s e d to modify t h e i m a g e c o n t r a s t . H e i n e , in
C H A N G I N G O P T I C A L D I C H R O I S M BY A N
collaboration with Leitz, p r o d u c e d a p h a s e con-
ELECTRIC FIELD
t r a s t effect w i t h a p a n c r a t i c c o n d e n s e r ( w i t h
A n electric field of a few volts o r i e n t a t e s t h e
v a r i a b l e focus) w h i c h p r o j e c t e d a t h i n r i n g of
D N A in t h e living t i s s u e . I t i n d u c e s o r i e n t a t i o n
l i g h t t a n g e n t i a l to t h e e d g e of t h e r e a r focal
of t h e f l u o r e s c e n c e of t h e fluorochromes
p l a n e (exit p u p i l ) of t h e o b j e c t i v e . T h i s is t h e
a t t a c h e d to t h e helix of t h e D N A .
f u n c t i o n of t h e v a r i o u s c o m p e n s a t o r s u s e d in
38 Instruments and techniques

L o c q u i n s h o w e d , in 1 9 5 3 , t h a t it is p o s s i b l e t o
b u i l d a p u p i l l a r y m o d i f i e r c a p a b l e of f u n c t i o n i n g
in n a t u r a l light w i t h o u t discontinuity to o b t a i n
p h a s e c o n t r a s t w i t h o u t fringes. T h e modulator
c o n s i s t s of a F a b r y - P e r r o t filter w h i c h m o d u l a t e s
l i g h t , b o t h in a m p l i t u d e a n d p h a s e , p r o g r e s s i v e -
ly w i t h i n c r e a s i n g d i s t a n c e f r o m t h e o p t i c a l a x i s .
T w o o t h e r less c o m p l e t e e a r l i e r a p p l i a n c e s are
known: the objective with a frosted internal
s c r e e n of S p i e r e r a n d L o c q u i n a n d t h e a n o p t e r a l
s y s t e m of W i l s k a i n its o r i g i n a l f o r m . T h e s e t w o
appliances could only m o d u l a t e amplitudes.
F i g u r e 1.39. An excess thickness e in T h e f o l l o w i n g c h a r t lists t h e m a i n pupillary
an object of uniform (refractive) index, modulators, together with t h e n a m e s of t h e i r
introduces a phase difference of phase
inventors.
displacement t. The direction of the
light is from left to right Diaphragms suitable for natural light
With discontinuous structure
A w (a) Central dark field, internal reflector objective (Spierer).
(b) Annular attenuator, anopteral contrast (Wilska).
(c) Annular phase plates, phase contrast (Zernike).
(d) Interference phase plates, colour contrast (Locquin,
Varicolor).
(e) Double phase plate, coloured colour contrast (Zernike).
(f) Dispersion phase plates, colour contrast (Interphako).
With continuous structure
(a) Vignetted central screen, Spierer-Locquin objective.
(b) Fabry-Perrot filter on a carriage with mirror (Dyson).
(c) Fabry-Perrot filter mounted on the front of the objective
or on the cover-slip, Locquin variable contrast (Mod-
ecla).
Modulators used with polarized light
With discontinuous structure
(a) Half-wave phase plate, Locquin variable phase con-
trast.
(b) Compound phase plate, Osterberg variable contrast.
(c) Rotating phase plate, Taylor variable contrast.
With continuous structure
(a) Accessories for the polarizing microscope: quartz, sensi-
tive stains, red first class; quarter-wave mica according
to Senarmont, for circular polarization; corner quartz,
1st and 3rd class; revolving mica of Brace-Khler,
one-tenth to one-thirtieth wave; Eringhaus revolving
compensator quartz or spar, 7 classes; Babinet compen-
sator; half-light plates; half-wave plates; wave plates;
oscillating magnesium fluoride plates; oscillating calcite
plates, 30 classes.
w=u+v
(b) Accessories for interference microscopes: Mach-Zender
Figure 1.40. Interference between two compensator (Interphako); Wollaston compensator
waves u and and their resultant w in ( C N R S - N o m a r s k i ) ; Jamin-LebedefT compensator
three cases. Topphase coincidence; (Leitz); Savart, Blaisse or Franon compensator;
bottomphase divergence; Babinet-Soleil compensator (Philpot); semi-wave com-
centrepartial phase shift pensator + Mac de Lepinay-Locquin.

p o l a r i z a t i o n . I t is a l s o t h e f u n c t i o n of t h e p h a s e
Interference m i c r o s c o p e s
p l a t e s , a l t h o u g h in t h i s c a s e t h e effect is m o r e I n p r i n c i p l e , t h e r e a r e t h r e e t y p e s of i n t e r f e r e n c e
localized. I n t h e latter case, t h e d i s c o n t i n u i t y in microscopes, the split-wave interference micro-
the phase plate introduces a secondary effect scope, the two-wave interference microscope
w h i c h is r e s p o n s i b l e for t h e h a l o surrounding a n d the multiple-wave interference microscopes
t h e i m a g e , i n a d d i t i o n t o t h e c o n t r a s t effect. (Figures 1.39 a n d 1.40).
Chief instrumental methods 39

F i g u r e 1.41. Light path through three interferometers


used in transmission in interference microscopy.
P, polarizer; W, Wollaston; , half-wave plate;
PE, object plane; O b , objective; C, condenser;
A, analyser; K, quartz plate; BE 0b> objective focal plane;
B E C, condenser focal plane. The dots along the light
path indicate that the direction of the vibrations of
polarized light is perpendicular to the plane of the paper,
while the lines show it to be parallel

Smith Nomarski

F i g u r e 1.42. Nomarski interference


contrast image of the diatom Navicula
clavata Gregory (Zeiss Ultraphot
objective Planapo 40, magnification
approx. X1000, Polaroid PN55 film,
photographed by P. Fusey,
Cryptogamy Laboratory of the
National Museum of Natural History,
Paris)

F i g u r e 1.43. Diagram showing the


setting up of Nomarski interference in
a B B T microscope. Pu P2, polarizer;
QDJ QCJ Wollaston units; q, Wollaston
composite with variable splitting
(BBT, Paris)
40 Instruments and techniques

T h e first t y p e , t h e o n e - w a v e i n t e r f e r e n c e m i n u t e i n t e r n a l d e t a i l s s u c h as n u c l e i , n u c l e o l i ,
m i c r o s c o p e , is n o t g e n e r a l l y k n o w n u n d e r this m i t o c h o n d r i a , etc. F. Zernike was a w a r d e d the
n a m e , b u t is called a p h a s e c o n t r a s t m i c r o s c o p e . N o b e l p r i z e for t h i s d i s c o v e r y (Figure 1.44).
Nevertheless, they certainly are interference I n o r d e r to u n d e r s t a n d p h a s e c o n t r a s t , t h e
m i c r o s c o p e s , since t h e i n c i d e n t light w a v e is reader should again read the relevant passage,
s e p a r a t e d i n t o t w o r a y s , o n e n o t modified b y t h e in S e c t i o n 1.1, o n t h e f u n d a m e n t a l m e c h a n i s m
o b j e c t , t h e o t h e r modified b y it. T h e final i m a g e of t h e f o r m a t i o n of i m a g e s u n d e r a m i c r o s c o p e ,
is o n l y o b t a i n e d b y i n t e r f e r e n c e r e c o m b i n a t i o n d i s c o v e r e d b y A b b e ( p . 6 ) . O b j e c t s seen u n d e r a
of t h e s e t w o r a y s o n e of w h i c h h a s b e c o m e
s u i t a b l y o u t of p h a s e w i t h t h e o t h e r . I n m o s t
instruments the phase displacement introduced
by the phase plate remains permanent.
T h e s e c o n d c a t e g o r y , t h e t w o - w a v e interfer-
ence microscopes, are true interference micro-
s c o p e s . Before p a s s i n g t h r o u g h t h e object, t h e
i n c i d e n t b e a m of i l l u m i n a t i n g light is split i n t o
t w o b e a m s close to e a c h o t h e r b y o p t i c a l s y s t e m s
of v a r y i n g d e s i g n s a n d this s p l i t t i n g c a n g e n e r a l - F i g u r e 1.44. Change in phase and
amplitude of a beam on passing
ly b e v a r i e d b y t h e o p e r a t o r ; a s e c o n d o p t i c a l through the object (Ob), then
s y s t e m , s y m m e t r i c a l w i t h t h e first, b u t l o c a t e d through the phase plate (Ph).
after t h e object, r e c o m b i n e s t h e t w o split b e a m s , Leftdirect beam;
g i v i n g rise to i n t e r f e r e n c e to form t h e i m a g e a rightdiffracted beam
{Figures 1.41-1.43). microscope can be divided into two categories
T h e t h i r d c a t e g o r y , t h e so-called m u l t i p l e - coloured or d a r k objects a b s o r b a certain
w a v e i n t e r f e r e n c e m i c r o s c o p e , is s e l d o m u s e d in a m o u n t of i n c i d e n t light a n d t h u s modify d e n s i -
b i o l o g i c a l m i c r o s c o p y . O n t h e o t h e r h a n d , it ty a n d c o l o u r . T h e s e a r e k n o w n a s a m p l i t u d e
finds w i d e s p r e a d a p p l i c a t i o n in m e t a l l u r g y for o b j e c t s . T h e o t h e r t y p e c o n s i s t s of c o l o u r l e s s
m e a s u r i n g d e p t h s of d e t a i l s . T h i s t y p e a c t s as a o b j e c t s , g e n e r a l l y of slight t h i c k n e s s , t h e c o n -
m i c r o i n t e r f e r o m e t e r w i t h m u l t i p l e fringes, in t o u r s a n d d e t a i l s of w h i c h a r e clearly o b s e r v e d
w h i c h a f r a m e of p a r a l l e l fringes, t h e m s e l v e s o n l y in a d a r k field. T h e s e a r e k n o w n a s p h a s e
m o d i f i e d b y t h e s t r u c t u r e of t h e object, is s u p e r - objects. Physicists have established that there
i m p o s e d o n t h e i m a g e of t h e object. exists a f u n d a m e n t a l difference b e t w e e n t h e s e
I n 1948, M . L o c q u i n w a s t h e first to d e v e l o p a t w o c a t e g o r i e s of o b j e c t s w h i c h is to b e f o u n d in
d e v i c e for o b s e r v a t i o n b y i n t e r f e r e n c e m i c r o s - t h e focal p l a n e of t h e object; it involves t h e
c o p y , b y e n c l o s i n g t h e o b j e c t in a F a b r y - P e r r o t r e s p e c t i v e p h a s e s t a t e s of light r a y s diffracted b y
i n t e r f e r o m e t e r ; it c o n s i s t e d of a t h i n s e m i - t h e o b j e c t in r e l a t i o n to d i r e c t r a y s , n o t m o d i f i e d
t r a n s p a r e n t l a y e r d e p o s i t e d o n a p l a t e a n d of a n b y t h e o b j e c t . F o r p h a s e o b j e c t s t h e r e is a p h a s e
u l t r a f i n e p l a s t i c film c o v e r i n g t h e object, w h i c h shift of / 4 , w h i c h d o e s n o t o c c u r w i t h a m p l i -
is also s e m i - m e t a l l i z e d . A s p e c i m e n m o u n t e d in t u d e objects. F r o m this, Zernike obtained the
t h i s w a y o n t h e s t a g e of a n o r d i n a r y m i c r o s c o p e i d e a of c o m p e n s a t i n g this p h a s e shift t h r o u g h
produces an interference contrast image either t h e o b j e c t i v e b y t h e m o d i f y i n g effect of a p h a s e
w i t h a p l a i n t e x t u r e , if t h e o b j e c t is v e r y t h i n , o r plate which, by compensation, causes the phase
w i t h m u l t i p l e fringes if it is t h i c k e r . T h i s d e v i c e s t a t e to r e v e r t to t h a t w h i c h w o u l d b e g i v e n b y
h a s t h e a d v a n t a g e t h a t it c a n b e u s e d w i t h a n a n ' a m p l i t u d e object' a n d thus converts the
ordinary microscope with a bright background. p h a s e c o n t r a s t to a m p l i t u d e c o n t r a s t . T h e t r a n s -
p a r e n t s p e c i m e n is c o n t r a s t e d w i t h o u t t h e u s e of
s t a i n s . I n p r a c t i c e , p h a s e p l a t e s a r e of a n n u l a r
Phase contrast s h a p e . T h e y a r e p l a c e d w i t h i n t h e i n t e r n a l focal
In the immediate post-war years, a great up- p l a n e of t h e o b j e c t i v e s . I n t h e t u b e of a s p e c i a l
h e a v a l in t h e t e c h n i q u e s of m i c r o s c o p i s t s w a s condenser, the designer has placed two transpar-
c a u s e d b y t h e d i s c o v e r y t h a t it w a s p o s s i b l e to ent illuminating rings which can be centred
o b s e r v e live s p e c i m e n s , e v e n e x t r e m e l y t h i n a n d paired with each phase plate. As the phase
ones, w i t h o u t applying lethal stains a n d yet p l a t e s a r e also m a d e a b s o r b e n t to r e d u c e t h e
o b t a i n a c l e a r a n d c o n t r a s t e d i m a g e of t h e m o s t i n t e n s i t y of t h e d i r e c t light, a n a u x i l i a r y s y s t e m
41

F i g u r e 1.45. Section of a phase contrast


microscope, showing the annular
diaphragm under the condenser and the
phase plate paired in the objective focal
plane (Wild-Heerbrugg)

Figure 1.46. Centring of the ring of light


on the phase plate. View through the
auxiliary centring microscope. The clear
image is made to coincide with the phase .
plate by operating the diaphragm centring
screws of the condenser. If the image is not F i g u r e 1.47. Light path in a phase
clear, the object is probably diffusing the contrast microscope. The diffracted light,
light too strongly and is not suitable for grey, is separated from the direct light,
examination under phase contrast white (Leitz-Wetzlar)
42 Instruments and techniques

m a k e s it p o s s i b l e to a d j u s t o n e o n t h e o t h e r b y d i r e c t i o n s of p r o p a g a t i o n a n d r o t a t i o n a r e n u m -
centring the d i a p h r a g m a n d the phase plate b e r e d s t a r t i n g from a n a s s u m e d r a y a l w a y s
w i t h t h e i n s t r u m e n t in p o s i t i o n for c a r r y i n g o u t c o m i n g t o w a r d s t h e o b s e r v e r , a s in a m i c r o -
o b s e r v a t i o n s (Figures 145-1.47). scope.
W h e n using a phase contrast microscope, the N a t u r a l light, to w h i c h w e a r e e x p o s e d d u r i n g
following m u s t b e b o r n e in m i n d . T h e s p e c i m e n s t h e d a y , like t h e light p r o d u c e d b y a m i c r o s c o p e
to b e e x a m i n e d m u s t b e s t a i n e d slightly o r n o t a t l a m p , v i b r a t e s d u r i n g its p r o p a g a t i o n in r a n d o m
all; t h e y m u s t n o t c o n t a i n a n y t h i c k m e m b r a n e f a s h i o n in all d i r e c t i o n s p e r p e n d i c u l a r to its
o r b e thick t h e m s e l v e s ; t h e y m u s t n o t c o v e r t h e p a t h . I f t h i s l i g h t is reflected b y a p o l i s h e d
w h o l e of t h e field so t h a t a n a p p r e c i a b l e p a r t of s u r f a c e , o r if it p a s s e s t h r o u g h p o l a r i z e r s , it is
t h e i n c i d e n t light d o e s n o t p a s s t h r o u g h t h e s a i d to b e p o l a r i z e d . T h e r e a r e s e v e r a l t y p e s of
o b j e c t . T h e o p t i c a l surface of b o t h t h e m i c r o - p o l a r i z a t i o n : r e c t i l i n e a r p o l a r i z a t i o n in w h i c h
s c o p e a n d of t h e p r e p a r a t i o n m u s t b e v e r y c l e a n . v i b r a t i o n s o n l y o c c u r in t h e o n e p l a n e ( t h e p l a n e
T h e light source m u s t be m o r e intense t h a n that of p o l a r i z a t i o n ) , c i r c u l a r p o l a r i z a t i o n in w h i c h
u s e d for t h e b r i g h t field b e c a u s e of t h e a n n u l a r t h e v i b r a t i n g v e c t o r r o t a t e s w h i l e a d v a n c i n g in
r e d u c t i o n of t h e i n c i d e n t light a n d especially t h e f o r m of a helix, a n d elliptic p o l a r i z a t i o n in
b e c a u s e of a b s o r p t i o n b y t h e p h a s e p l a t e . w h i c h t h e p r o j e c t i o n of t h e v e c t o r is elliptical
T h e r e a r e t h r e e b a s i c t y p e s of p h a s e p l a t e , in a p l a n e p e r p e n d i c u l a r to t h e d i r e c t i o n of
with numerous absorption variants: p r o p a g a t i o n of t h e l i g h t . I n t h e last t w o c a s e s ,
t h e r e c a n b e t w o d i r e c t i o n s of r o t a t i o n , to t h e
(1) ' P o s i t i v e ' p h a s e p l a t e s , w h i c h p r o d u c e a
r i g h t (clockwise) o r to t h e left ( a n t i - c l o c k w i s e )
d a r k i m a g e o n a g r e y field from a n o b j e c t
(Figures 1.48 a n d 1.49).
h a v i n g a g r e a t e r refractive i n d e x t h a n t h e
A p o l a r i z e r m u s t b e v i e w e d o b l i q u e l y to t h e
mounting medium.
5 g l a s s s u r f a c e to d e t e r m i n e t h e a x i s . T h e axis is
(2) ' N e g a t i v e p h a s e p l a t e s , w h i c h p r o d u c e
v e r t i c a l if reflections w h i c h a r e p o l a r i z e d w i t h
f r o m t h e s a m e o b j e c t a n i m a g e close to t h a t
r e s p e c t to t h e glass surface a r e e x t i n g u i s h e d .
in a d a r k field b u t r i c h e r in m e a n v a l u e s a n d
E x t i n c t i o n o c c u r s w h e n t h e a x e s of t h e t w o
in d e t a i l .
p o l a r i z e r s a r e c r o s s e d . T h e d e g r e e of c o m p l e t e -
(3) I n t e r f e r e n c e p h a s e p l a t e s ( L o c q u i n , 1950),
n e s s of this e x t i n c t i o n i n c r e a s e s w i t h t h e i m -
which produce colour contrast by super-
p r o v e d q u a l i t y of t h e p o l a r i z e r s (Figure 1.50).
p o s i t i o n of t h e s e t w o effects in t w o different
A s p e c i m e n is s a i d to b e b i r f r i n g e n t if, w h e n
w a v e l e n g t h r e g i o n s . T h i s last t y p e h a s b e e n
p l a c e d b e t w e e n t w o c r o s s e d p o l a r i z e r s , it p a r -
superseded by two-wave interference micro-
tially r e - e s t a b l i s h e s t h e light. If o n e p e r f o r m s t h e
scopes.
e x p e r i m e n t w i t h a c e l l o p h a n e film, t h e light r a y s
P o s i t i v e p h a s e p l a t e s a r e b y far t h e m o s t c o m - a r e r e f r a c t e d b y t h e object in t w o different w a y s ;
monly used. T h e image produced by t h e m dis- t h e n o r m a l r a y follows D e s c a r t e s ' l a w of refrac-
plays a characteristic halo which surrounds t i o n , sin i = sin r; t h e s e c o n d o n e t h e
e v e r y object. T h i s h a l o d o e s n o t exist in r e a l e x t r a o r d i n a r y r a y v i b r a t e s a t r i g h t a n g l e s to
interference contrast. t h e p r e v i o u s o n e a n d is p r o p a g a t e d a t a different
velocity, which m e a n s that birfringent subs-
t a n c e s h a v e t w o refractive i n d i c e s . F o r m e r l y ,
Polarizing microscope p o l a r i z e r s w e r e m a d e of I c e l a n d s p a r s u i t a b l y
c u t to e l i m i n a t e t h e n o r m a l r a y b y t o t a l reflec-
I t is n o t n e c e s s a r y to s t u d y c r y s t a l l o g r a p h y in t i o n followed b y a b s o r p t i o n . A t p r e s e n t , t h e y a r e
o r d e r to s t u d y b i o l o g i c a l s t r u c t u r e s u n d e r a p r o d u c e d a s n e u t r a l g r e y films w h i c h a b s o r b t h e
p o l a r i z i n g m i c r o s c o p e , e x c e p t in cases in w h i c h n o r m a l r a y in t h e i r o w n t h i c k n e s s . T h e y a r e
p a r t i c u l a r i n t e r e s t a t t a c h e s to c r y s t a l l i n e i n c l u - k n o w n a s p o l a r i z i n g discs o r P o l a r o i d s , t h e
s i o n s ; n o r is t h e r e a n y n e e d for a n y v e r y d e t a i l e d n a m e g i v e n to t h e m b y t h e b i g g e s t m a n u f a c t u r -
k n o w l e d g e of o p t i c s . e r s (Figure 1.51).
L i g h t is p a r t l y of w a v e a n d p a r t l y of c o r p u s c u - U s u a l l y o n e c a n o b s e r v e a r e a l i m a g e of a n
lar nature. For the present we are concerned o b j e c t in m i c r o s c o p y . T h i s is t h e o r t h o s c o p i c
with the wave aspect. m e t h o d of o b s e r v a t i o n . W h e n v i e w i n g t h e focal
A c c o r d i n g to g e n e r a l c o n v e n t i o n , a n d c o n - p l a n e of t h e o b j e c t i v e w i t h a c e n t r i n g lens to
t r a r y to w h a t is c u s t o m a r y in m e c h a n i c s , t h e p e r f o r m A b b e ' s e x p e r i m e n t s o r to c e n t r e t h e
43

u m
I '
F i g u r e 1.48. Double refraction. Simple
refraction (A), double refraction (B).
i incident ray, r and r' refracted rays
A

F i g u r e 1.49. Polarization of light. Left to rightnatural


light vibrates in all directions; after passing through a
polarizer, it only vibrates in the plane; a perpendicular
crossed analyser stops it completely. The field is dark
(Wild-Heerbrugg)

Figure 1.50. Diagram illustrating the


function of polarizing discs using a grid. 1,
parallel; 2, crossed

-
F i g u r e 1.51. Illumination of a double
refracting object between two crossed
polarizing discs. Left to rightsource,
vertical polarizer, horizontal analyser;
topwithout object; bottomwith a
birfringent object. The birfringent
object appears bright on a dark field
(Wild-Heerbrugg)
44 Instruments and techniques

l i g h t of a p h a s e p l a t e , c o n o s c o p i c o b s e r v a t i o n is p r o p e r t i e s or, if t h e s e a r e k n o w n , to d e t e r m i n e o r
k n o w n incorrectly as observation u n d e r con- c h e c k t h e n a t u r e of t h e object. T h e p o l a r i z i n g
verging light. T h i s conoscopic observation m i c r o s c o p e is m a i n l y u s e d in t h e s t u d y of t h i n
m a k e s it p o s s i b l e to view h o l o g r a p h i c interfer- s e c t i o n s of r o c k s . T h e n a t u r e of t h e c o n s t i t u e n t
e n c e figures w h i c h a r e p r o d u c e d in t h e focal m i n e r a l s c a n b e d e t e r m i n e d from t h e c r y s t a l
p l a n e of t h e o b j e c t i v e . I n c o r p o r a t i o n of a r e - s t r u c t u r e s a n d from t h e i r b r i g h t c o l o u r s b e t w e e n
t r a c t a b l e l e n s , k n o w n as B e r t r a n d ' s l e n s , in a c r o s s e d p o l a r o i d s . T h i s s t u d y d o e s n o t fall w i t h -
p o l a r i z i n g m i c r o s c o p e m a k e s it m u c h e a s i e r to in o u r s p h e r e , b u t p o l a r i z i n g m i c r o s c o p e s a r e
carry out observations than when using the v e r y useful in a n i m a l a n d p l a n t h i s t o l o g y (Figure
a b o v e - m e n t i o n e d auxiliary lens. 1.52).
A n y l i n e a r p o l a r i z e d light c a n b e r e s o l v e d i n t o
two vectors at right angles, which are the two
a x e s of t h e p o l a r o i d s in t h e m i c r o s c o p e . T h e s e
two vectors are expressed by the formulae
a p cos
b = p sin oc

where denotes the angle between the vector


a n d t h e p l a n e of t h e p o l a r i z e r ; it c a n b e seen t h a t
if all t h e light v i b r a t e s a c c o r d i n g to a, n o t h i n g
will h a p p e n to b if oc = 0. O n t h e o t h e r h a n d , if
oc 0, t h e t r a n s m i t t e d light i n t e n s i t y is r e p r e -
sented by the formula

/An = Ip cos 2oc = V2 Io cos 2oc

since a n y polarizer halves the incident intensity


d u r i n g polarization by eliminating the n o r m a l
ray. A birfringent object between two polariz-
e r s , a s m e n t i o n e d a b o v e , h a s t w o refractive
indices a n d two rays a n d hence the two waves
p a s s t h r o u g h a t different velocities, o n e r e t a r d e d
r e l a t i v e to t h e o t h e r . O n l e a v i n g t h e b i r f r i n g e n t
or anisotropic m e d i u m , they proceed indepen-
dently without changing their delay, passing
t h r o u g h t h e a n a l y s e r , w h i c h selects t h e t w o
c o m p o n e n t s a c c o r d i n g to b. T h e s e t w o c o m p o -
n e n t s will t h e n b e a b l e to form t h e i m a g e b y
i n t e r f e r e n c e , a s t h e y v i b r a t e in t h e s a m e p l a n e .
I n o r d e r to facilitate q u a n t i t a t i v e o b s e r v a t i o n
a n d m e a s u r e m e n t s , t h e p o l a r i z i n g m i c r o s c o p e is
p r o v i d e d in its t u b e w i t h a n o b l i q u e slot a t a 45
a n g l e to t h e p o l a r o i d s i n t o w h i c h fixed o r m o b i l e
F i g u r e 1.52. Polarizing microscope for practical work,
c o m p e n s a t o r s m a y b e i n s e r t e d . M o r e o v e r , as t h e -POL, BBT. Observe the rotating stage divided with a
s t a g e of t h e m i c r o s c o p e revolves a n d c a n b e vernier (a standard accessory), objectives which can be
c e n t r e d , t h e s p e c i m e n c a n b e m a d e to r o t a t e o n centred individually on the revolving nosepiece, and the
itself w h i l e b e i n g k e p t in t h e field. O n s o m e carriage for auxiliary slides (BBT, Paris)

s t a n d s , t h e p o l a r s a r e j o i n e d b y a b a r ; in t h i s
case they rotate together while the object re- U n f o r t u n a t e l y t h i s m e t h o d of r e s e a r c h is sel-
m a i n s fixed in p o s i t i o n . d o m u s e d . Y e t c e r t a i n f e a t u r e s of tissues c a n b e
d e m o n s t r a t e d with a sharpness unrivalled by
U S E O F A POLARIZING M I C R O S C O P E m e t h o d s of s t a i n i n g o r i m p r e g n a t i o n . I t c o u l d
A p o l a r i z i n g m i c r o s c o p e gives i n f o r m a t i o n o n even reveal structural details, not otherwise
t h e b i r e f r i n g e n c e of b o d i e s . I t c a n t h u s b e u s e d d e t e c t e d b y t h e i r r e f r a c t i o n , a b s o r p t i o n o r col-
t o d e t e r m i n e e i t h e r t h e n a t u r e of t h e s e o p t i c a l o u r or by staining.
Chief instrumental methods 45

PRECAUTIONS microscope does not have a revolving stage, one


C o n s i d e r a b l e losses of light t a k e p l a c e , a s a can rotate the preparation by h a n d . T h i s does
r e s u l t of a b s o r p t i o n in t h e t w o p o l a r i z i n g d i s c s . n o t p r e s e n t a n y g r e a t difficulties a t m o d e r a t e
L i g h t r a y s a r e split t w i c e b y a b i r f r i n g e n t o b j e c t magnifications and with some practice.
b e t w e e n c r o s s e d p o l a r i z e d d i s c s , a n d so t h e M o s t r e v o l v i n g s t a g e s w h i c h a r e s u p p o s e d to
o b s e r v e r n e v e r receives m o r e t h a n o n e - q u a r t e r b e c e n t r a b l e a r e v e r y difficult to c e n t r e e x a c t l y .
of t h e i n c i d e n t light. I t is n e c e s s a r y to g u a r d First, the stage should be centred approximately
a g a i n s t a n y e x t r a n e o u s light, w h e n m a k i n g w i t h t h e a d j u s t i n g s c r e w . A slide, o n w h i c h a
o b s e r v a t i o n s u n d e r p o l a r i z e d light. T h e r e f o r e , c r o s s o r a s m a l l circle w i t h a d i a m e t e r of a b o u t
t h e u s e of t h e s c r e e n s d i s c u s s e d e a r l i e r is s t r o n g - 1 m m h a s been d r a w n with ink or with a di-
ly r e c o m m e n d e d . A l a r g e s h e e t of b l a c k c a r d - a m o n d , is e x a m i n e d a t v e r y low m a g n i f i c a t i o n .
b o a r d is t h e b e s t t y p e o f s c r e e n , sufficiently h i g h T h i s o b j e c t is p l a c e d d i r e c t l y in t h e c e n t r e of t h e
t o c o v e r t h e w h o l e of t h e o b s e r v e r ' s h e a d , a n d field a n d c l a m p e d w i t h t h e clips o r t h e c a r r i a g e ,
h a v i n g t h e l o w e r p o r t i o n c u t o u t for t h e r a y s a n d t h e s t a g e is t h e n r o t a t e d slowly. T h e o b j e c t
from t h e light s o u r c e to p a s s to t h e m i r r o r , if t h e p a s s e s o u t s i d e t h e field of vision; it m u s t b e k e p t
s o u r c e is n o t i n c o r p o r a t e d in t h e i n s t r u m e n t . w i t h i n it b y m o v i n g t h e o b j e c t b y h a n d , c o m b i n -
C a r e m u s t b e t a k e n t h a t t h e slides a n d c o v e r - i n g t h e s e m o v e m e n t s w i t h t h o s e of t h e c e n t r i n g
slips a r e c o m p l e t e l y free from b i r e f r i n g e n c e , s c r e w . T h e s t a g e is m a d e to p e r f o r m a c o m p l e t e
w h e n carrying out critical observations o n o b - revolution, by trial a n d error, w i t h o u t allowing
j e c t s of l o w b i r e f r i n g e n c e . F o r t h i s p u r p o s e , t h e y t h e o b j e c t t o p a s s o u t s i d e t h e field of vision. O n e
c a n b e e x a m i n e d w i t h a sensitive g y p s u m o r repeats the centring at higher magnification.
q u a r t z p l a t e . I n t e r f e r e n c e d u e to t h e b i r e f r i n g - T h e c e n t r i n g s c r e w is n o t t o u c h e d a g a i n from
e n c e of t h e e y e p i e c e o r of t h e o b j e c t i v e is m u c h then onwards.
less c o m m o n s i n c e t h o s e a r e e s p e c i a l l y s e l e c t e d W i t h the stage centred a n d the object rotating
by the manufacturer. b e t w e e n t h e c r o s s e d p o l a r o i d s , t w o different
O n e must examine separately a dry specimen c a s e s m a y c o n f r o n t t h e o b s e r v e r . If t h e object is
a n d a n o t h e r i m m e r s e d in C a n a d a b a l s a m , to e x t i n g u i s h e d four t i m e s a n d i l l u m i n a t e d four
study dried crystalline precipitates; the latter t i m e s in p o s i t i o n s a t 45 r e l a t i v e to o n e a n o t h e r
c a u s e s i n t e r f e r e n c e c o l o u r s to s t a n d o u t . I n t h i s ( t h i s c a n b e verified w i t h t h e g r a d u a t e d circle of
c a s e , it is a d v a n t a g e o u s to m o u n t t h e s p e c i m e n t h e a n a l y s e r ) , it is e v i d e n t l y b i r f r i n g e n t . T h e
u n d e r a c o v e r - s l i p w i t h a v e r y s m a l l a m o u n t of i l l u m i n a t i o n m a y b e t o o w e a k for v e r y t h i n
b a l s a m in s u c h a w a y t h a t a p o r t i o n of t h e o b j e c t s o r t h o s e of v e r y low a n i s o t r o p y . If t h e
c r y s t a l l i n e m a t e r i a l is in t h e air. field r e m a i n s d a r k d u r i n g a c o m p l e t e r e v o l u t i o n
of t h e s t a g e , t h e r e a r e t h r e e possibilities:
BIREFRINGENCE EXAMINATIONS
(1) T h e o b j e c t is b i r f r i n g e n t b u t p o o r l y
T h e p o l a r i z e r is set u p , t h e o b j e c t is p l a c e d o n
o r i e n t e d . If its axis c o i n c i d e s w i t h t h a t of t h e
t h e s t a g e a n d t h e a n a l y s e r is t h e n p u t i n t o
m i c r o s c o p e , it will b e h a v e like a n i s o t r o p i c
p o s i t i o n . T h e i n s t r u m e n t s h o u l d b e focused ex-
o b j e c t . I n t h i s c a s e , t h e o b j e c t is m o v e d o r
actly between the parallel polarizing discs, a n d
a n o t h e r s p e c i m e n is p r e p a r e d .
t h e a n a l y s e r is t h e n t u r n e d t h r o u g h 90 so t h a t
(2) T h e o b j e c t is fully i s o t r o p i c ; t h e c h a n g e in
t h e p o l a r i z i n g d i s c s a r e c r o s s e d . I t is c o n v e n i e n t
orientation a n d the g y p s u m plate leave the
to m a k e t h e p l a n e of v i b r a t i o n of t h e a n a l y s e r
field c o m p l e t e l y d a r k in all p o s i t i o n s .
c o i n c i d e w i t h t h e s a g i t t a l p l a n e of t h e m i c r o -
(3) T h e o b j e c t is too w e a k l y b i r f r i n g e n t to
s c o p e ; t h a t of t h e p o l a r i z e r w h i c h is p e r p e n d i c u -
p r o d u c e i l l u m i n a t i o n . I n this c a s e , e x a m i n a -
l a r to t h e f o r m e r , is t h e n p a r a l l e l to t h e f r o n t a l
t i o n is c a r r i e d o u t w i t h a sensitive q u a r t z o r
p l a n e of t h e m i c r o s c o p e .
gypsum plate.
W i t h t h e p o l a r o i d s c r o s s e d , t h e o b j e c t is r o -
t a t e d slowly w i t h i n t h e p l a n e of t h e s t a g e a b o u t D E T E R M I N A T I O N OF T H E SIGN OF T H E
t h e o p t i c a l axis of t h e m i c r o s c o p e . H e r e , t w o BIREFRINGENCE
c a s e s m a y a r i s e . I f t h e m i c r o s c o p e is e q u i p p e d A s s t a t e d a b o v e , t h e g y p s u m p l a t e is u s e d w h i c h
with a revolving stage, this m u s t be centred gives first-order r e d . B i r e f r i n g e n c e is p o s i t i v e o r
e x a c t l y ; it c a n o n l y b e u s e d if t h i s c o n d i t i o n is n e g a t i v e , d e p e n d i n g o n w h e t h e r t h e velocity of
m e t . O t h e r w i s e t h e o b j e c t will p a s s o u t s i d e t h e t h e o r d i n a r y r a y is g r e a t e r (4-) o r s m a l l e r ( )
field w h i l e t h e s t a g e is b e i n g r o t a t e d . If t h e t h a n t h a t of t h e e x t r a o r d i n a r y r a y . I n t h e first
46 Instruments and techniques

c a s e , t h e c o l o u r i n c r e a s e s ( t o w a r d s b l u e ) , in t h e BIREFRINGENT SUBSTANCES
s e c o n d it d e c r e a s e s ( t o w a r d s y e l l o w ) . B i r e f r i n g e n c e m a y b e d u e to v a r i o u s p h y s i c a l
T h e sign of a u n i a x i a l c r y s t a l c a n b e d e t e r - c a u s e s , of w h i c h t h e m o s t i m p o r t a n t a r e :
m i n e d in c o n v e r g e n t light u s i n g a q u a r t e r - w a v e
(1) T h e c r y s t a l l i n e s t r u c t u r e , i.e. o r i e n t a t i o n of
m i c a d i s c . T h e r i n g s in t h e q u a d r a n t s l y i n g
t h e m o l e c u l e s in a t h r e e - d i m e n s i o n a l l a t t i c e .
b e t w e e n t h e a r m s of t h e cross a r e d i s p l a c e d a n d
All c r y s t a l s e x c e p t t h o s e of t h e c u b i c s y s t e m
t w o b l a c k s p o t s a p p e a r n e a r t h e c e n t r e . If t h e s e
are birfringent.
s p o t s a r e p a r a l l e l to t h e b l a c k a r r o w of t h e m i c a
(2) T h e m i c e l l a r s t r u c t u r e of colloidal s u b s -
p l a t e , t h e c r y s t a l h a s a n e g a t i v e sign, w h i l e if t h e
t a n c e s . O r i e n t a t i o n of t h e micelles m a y , in
c o m b i n a t i o n of t h e t w o s p o t s lies p e r p e n d i c u l a r
c e r t a i n c a s e s , give rise to s e m i - i s o t r o p y , i.e.
to t h e a r r o w , t h e sign of t h e c r y s t a l is p o s i t i v e .
an arrangement intermediate between the
c r y s t a l l i n e a n d a m o r p h o u s s t a t e s , as in col-
D E T E R M I N A T I O N OF VERY WEAK l a g e n fibres.
BIREFRINGENCE
(3) M o l e c u l a r s t r a i n , w h i c h c a u s e s s o m e s u b s -
F i r s t , c a r e m u s t b e t a k e n to e n s u r e m a x i m u m
t a n c e s to b e c o m e b i r f r i n g e n t w h e n t h e y a r e
i l l u m i n a t i o n of t h e p r e p a r a t i o n b y u s i n g a h i g h
c o m p r e s s e d or stretched. Gelatin, glass,
i n t e n s i t y light s o u r c e a n d m a i n t a i n i n g t h e c o n -
s t r e t c h i n g a n d r e l a x a t i o n of b l o n d h a i r , e t c . ,
denser above the polarizer. In addition, either
a r e e x a m p l e s of t h i s .
t h e g y p s u m p l a t e is u s e d g i v i n g first-order r e d o r
(4) Diffraction p r o d u c e d b y v e r y finely r e t i c u -
t h e sensitive q u a r t z p l a t e gives s e c o n d - o r d e r
l a t e d s t r u c t u r e s t h a t p o l a r i z e light. Diffrac-
s e n s i t i v e n o . 1 violet. O b j e c t s h a v i n g a v e r y
tion birefringence disappears w h e n the ob-
w e a k b i r e f r i n g e n c e , w h i c h a r e h a r d l y visible o r
j e c t is p l a c e d in a m e d i u m h a v i n g t h e s a m e
invisible between crossed polarizing discs,
refractive i n d e x as itself.
n e v e r t h e l e s s affect c o l o u r s p r o v i d e d b y t h e s e
(5) B i r e f r i n g e n c e of flow is o b s e r v e d in s o m e
s e n s i t i v e p l a t e s , b e c a u s e of t h e e a s e w i t h w h i c h
l i q u i d o r v i s c o u s s u b s t a n c e s w h i c h a r e iso-
b i r f r i n g e n t s u b s t a n c e s modify t h e i r c o l o u r of
t r o p i c w h e n a t rest b u t b e c o m e a n i s o t r o p i c
polarization.
w h e n t h e y flow; for e x a m p l e , C a n a d a b a l -
T h e sensitive t i n t p l a t e s a r e p l a c e d b e n e a t h
s a m , purified v i r u s - p r o t e i n s , etc.
t h e a n a l y s e r , t h e m o u n t i n g of w h i c h m u s t b e
provided with a special indentation. T h e object
is r o t a t e d a n d t h e c h a n g e in c o l o u r is o b s e r v e d . PLEOCHROISM
W h e n t h e g y p s u m p l a t e is u s e d , t h e r e d t i n t T h e t e r m p l e o c h r o i s m d e n o t e s v a r i a t i o n s in
c h a n g e s e i t h e r to b l u e o r to yellow; w i t h t h e c o l o u r c h a r a c t e r i s t i c s for c o l o u r e d , b i r f r i n g e n t
q u a r t z p l a t e t h e c h a n g e is from violet to r e d o r to s u b s t a n c e s d u e n o t to i n t e r f e r e n c e b u t to u n -
i n d i g o . A m i c a p l a t e c a n also b e u s e d ; this is t h e e q u a l a b s o r p t i o n of p o l a r i z e d l i g h t in different
so-called q u a r t e r - w a v e m i c a , i.e. a p l a t e c u t in directions, d e p e n d i n g on the optical properties
such a way that the two polarized rays emerge of t h e s u b s t a n c e . T o o b s e r v e p l e o c h r o i s m , t h e
from t h e p l a t e w i t h a p h a s e difference of a a n a l y s e r is r e m o v e d a n d o n l y t h e p o l a r i z e r is
q u a r t e r - w a v e . T h e differences p r o d u c e d b y t h e u s e d . T h e o b j e c t is r o t a t e d as d e s c r i b e d a b o v e
o b j e c t a n d t h e m i c a a d d u p o r interfere w i t h a n d its c h a n g e s in c o l o u r a r e o b s e r v e d .
e a c h o t h e r , as in t h e c a s e of o t h e r sensitive S u b s t a n c e s in w h i c h it is p a r t i c u l a r l y e a s y to
plates, a n d the tint increases or decreases. study this p h e n o m e n o n include coloured
T h e sensitive t i n t p l a t e s m u s t b e o r i e n t e d in a carotene crystals, which during one complete
d i a g o n a l d i r e c t i o n w i t h r e s p e c t to t h e c r o s s e d r o t a t i o n a p p e a r t w i c e to b e p r a c t i c a l l y colourless
n i o i s , in o r d e r to o b t a i n t h e t i n t , so t h a t t h e axis a n d t w i c e to s h o w a n i n t e n s e r e d c o l o r a t i o n . I n
of l o w e r elasticity, i.e. t h e axis w h i c h c o r r e s - fact, c a r o t e n e c r y s t a l s t r a n s m i t p r a c t i c a l l y t h e
p o n d s to m a x i m u m refractive i n d e x , m a k e s a n w h o l e s p e c t r u m in o n e d i r e c t i o n b u t o n l y r e d
a n g l e of 45 w i t h t h e p l a n e of p o l a r i z a t i o n of t h e l i g h t in t h e o t h e r o n e . C e l l u l o s e m e m b r a n e s
p o l a r i z e r s . I n g e n e r a l , t h i s d i r e c t i o n is m a r k e d s t a i n e d violet b y i o d i n e - c o n t a i n i n g z i n c c h l o r -
o n t h e m o u n t i n g of t h e sensitive t i n t p l a t e . F o r ide, also clearly d e m o n s t r a t e pleochroism.
i n s t a n c e , in t h e c a s e of t h e g y p s u m p l a t e , a A p p l i c a t i o n of d i c h r o i s m in t h e u l t r a v i o l e t
definite p u r p l e t i n t m u s t b e o b t a i n e d b e t w e e n a n d t h e p o l a r i z a t i o n of fluorescent light h a v e
c r o s s e d p o l a r i z e r s , c h a n g i n g to g r e e n b e t w e e n e n a b l e d C . A . B a u d to i n v e s t i g a t e t h e s h a p e a n d
parallel polarizers. o r i e n t a t i o n of l a r g e m o l e c u l a r a g g r e g a t e s a n d of
Chief instrumental methods 47

m i c e l l e s , as well a s to s t u d y m o l e c u l a r con- strain-free optical system which has been tre-


figurations. a t e d carefully t o e l i m i n a t e all reflections. T o
verify w h e t h e r all t h e s e c o n d i t i o n s h a v e b e e n
fulfilled, o n e m u s t c h e c k w h e t h e r t h e field is
truly d a r k between crossed polarizing discs,
Study of anisotropy of biological tissues
even when using very intense illumination. In
u n d e r polarized light
c o n t r a s t to p o l a r i z a t i o n w h i c h d e t e c t s e x t i n c t i o n
I n g e n e r a l , t h e b i r e f r i n g e n c e of b i o l o g i c a l o b - o r m a x i m a o r m i n i m a of b r i l l i a n c e o n b o t h sides
j e c t s is less t h a n t h a t of m i n e r a l s . I n a d d i t i o n , it of e x t i n c t i o n , p h a s e d i c h r o i s m o n l y d e m o n s -
is f r e q u e n t l y v e r y difficult to find s t r u c t u r e s t r a t e s c o n t r a s t i n e q u a l i t i e s r e l a t i v e to t h e m e a n
w h i c h a r e sufficiently t h i c k to m a k e m e a s u r e - g r e y of t h e field in t h e a b s e n c e of a n object.
m e n t s . Therefore, very sensitive m e t h o d s m u s t T h e s e variations are not well-appreciated by the
be used. e y e , w h i c h h a s led B a r e r to u s e a h i g h l y sensitive
T h e elliptical c o m p e n s a t o r of B r a c e - K h l e r p h o t o m e t e r to c a r r y o u t his m e a s u r e m e n t s . T h e
( 1 9 2 1 ) is t h e e a s i e s t m e t h o d for m a k i n g a u t h o r h a s suggested combining the 'grey point'
m e a s u r e m e n t s , s i m p l y b y r e a d i n g a n a n g l e of m e t h o d of S. I n o u e (1951) w i t h t h a t of B a r e r ,
r o t a t i o n . T h e m i c a p l a t e of w h i c h it c o n s i s t s a n d p r o j e c t i n g t h e i m a g e of a c o m p o s i t e birfrin-
i n t r o d u c e s a d e l a y of t h e s a m e o r d e r of m a g n i - g e n t p l a t e , c o m p r i s i n g t w o c o m b i n e d 1/20 w a v e
t u d e a s t h e d e l a y in m a k i n g t h e m e a s u r e m e n t , plates, whose axes are at a small angle with each
u s u a l l y o n e - t w e n t i e t h o r o n e - t h i r t i e t h of t h e o t h e r . T h e i m a g e of t h i s p l a t e is p r o j e c t e d u n d e r
w a v e l e n g t h . T h e o b j e c t is o r i e n t e d b e t w e e n p o l a r i z e d l i g h t b y m e a n s of a n a c h r o m a t i c d e -
c r o s s e d p o l a r i z i n g d i s c s , a t a n a n g l e of 45 w i t h v i c e , w e l l - c o r r e c t e d for s p h e r i c a l a b e r r a t i o n , free
its p o s i t i o n of e x t i n c t i o n . If is t h e a n g l e from s t r a i n a n d w i t h o u t e m p l o y i n g a m i r r o r in
through which the compensator must be rotated t h e l i g h t p a t h (a m i r r o r w o u l d b r i n g a b o u t
to b r i n g a b o u t e x t i n c t i o n , t h e d e l a y i n t r o d u c e d p a r t i a l d e p o l a r i z a t i o n of t h e light b e a m ) . E v a -
b y t h e o b j e c t is g i v e n b y l u a t i o n of a c o n t r a s t v a l u e is s u b s t i t u t e d in t w o
h a l v e s of a n o b j e c t a c r o s s t h e line of s e p a r a t i o n
R = R0 sin 2 0 of t h e c o m p o s i t e L o c q u i n p l a t e , b y e v a l u a t i n g
w h e r e RQ is t h e c o m p e n s a t o r c o n s t a n t . W h e n t h e m a x i m u m c o n t r a s t d e v i a t i o n o r its e q u a l i t y ,
d i s p l a c e d slightly from t h e c o m p e n s a t e d p o s i - d e p e n d i n g o n w h e t h e r o n e is d e a l i n g w i t h a
tion, the object either a p p e a r s brighter or d a r k e r birfringent object or not.
t h a n t h e field, d e p e n d i n g o n w h e t h e r t h e p o s i - T h i s m e t h o d offers t h e a d v a n t a g e of e l i m i n a t -
t i o n is o n t h e a d d i t i o n o r s u b t r a c t i o n s i d e . T h e i n g s o u r c e s of e r r o r d u e to i m p e r f e c t i o n s of t h e
c o m p e n s a t o r c o n s t a n t , i.e. t h e d e l a y d u e to t h e optical system, a l t h o u g h these m a y persist as
p l a t e c o n s t i t u t i n g t h e c o m p e n s a t o r itself, is d e - f a c t o r s r a i s i n g t h e s e n s i t i v i t y t h r e s h o l d , w h i c h is
t e r m i n e d b y e x a m i n i n g a r e f e r e n c e c r y s t a l of less s e r i o u s . T h e c o m p o s i t e p l a t e of L o c q u i n c a n
k n o w n thickness a n d birefringence. o b v i o u s l y b e m a d e u p of t w o 45 h a l f - w a v e
Barer originally suggested replacing the a n a - p l a t e s , w h i c h give t w o h a l f - i m a g e s p o l a r i z e d a t
lyser of a p o l a r i z i n g m i c r o s c o p e b y a p h a s e r i g h t a n g l e s . W h e n u s i n g t h e elliptical c o m p e n -
c o n t r a s t o b j e c t i v e in o r d e r to i m p r o v e t h e effi- s a t o r s , o n e of t h e t w o i m a g e s is in t h e a d d i t i o n
c i e n c y of t h e i n s t r u m e n t . I n a p h a s e c o n t r a s t p o s i t i o n a n d t h e o t h e r o n e in a s u b t r a c t i o n
m i c r o s c o p e , t h e c o n t r a s t in t h e i m a g e d e p e n d s position, m u t u a l l y symmetrical a b o u t the posi-
o n t h e difference in t h e o p t i c a l p a t h s b e t w e e n t i o n of e x a c t c o m p e n s a t i o n . T h e c o m p e n s a t o r of
t h e o b j e c t a n d t h e s u r r o u n d i n g m e d i u m . If t h e M a c d e L p i n a y is a n a l o g o u s to a n elliptical
i n d e x d e p e n d s o n t h e o r i e n t a t i o n of t h e i n c i d e n t d o u b l e c o m p e n s a t o r of c o n t i n u o u s l y v a r i a b l e
p o l a r i z e d light, t h e c o n t r a s t v a r i e s in a c c o r d a n c e v a l u e . T h e w e a k e r t h e b i r e f r i n g e n c e s to b e m e a -
with this orientation. I n this m a n n e r we discov- s u r e d , t h e h i g h e r s h o u l d b e t h e a b s o r p t i o n of t h e
er w h a t I h a v e called ' p h a s e dichroism'. phase plate.
F o r t h e p r a c t i c a l a p p l i c a t i o n of t h i s m e t h o d T h e b i r e f r i n g e n c e of c r y s t a l l i n e s u b s t a n c e s is
we require a phase contrast microscope de- d e a l t w i t h in s p e c i a l i s t b o o k s o n m i n e r a l o g y a n d
s i g n e d like a p o l a r i z i n g m i c r o s c o p e , i.e. e q u i p - c r y s t a l l o g r a p h y . Biological b i r e f r i n g e n c e p r o -
p e d w i t h a d i v i d e d r e v o l v i n g s t a g e c a p a b l e of d u c e s n e w t y p e s of s t r u c t u r e s , u n k n o w n in in-
b e i n g c e n t r e d a n d in a d d i t i o n a n d t h i s is of organic chemistry. I n addition, crystalline sub-
particular importancewith a completely stances are homogeneous and impermeable and
48 Instruments and techniques

their birefringence does not d e p e n d on the tween two limiting layers but remain non-
m e d i u m in w h i c h t h e y a r e l o c a t e d ; t h e y p o s s e s s o r i e n t e d in t h e i r p l a n e .
a n intrinsic or p r i m a r y birefringence. Spherulitic structures display the phe-
Biological s t r u c t u r e s a r e h e t e r o g e n e o u s ; t h e i r n o m e n o n of t h e b l a c k c r o s s , w h i c h m a y b e
micelles or large macromolecules are substances negative or positive, d e p e n d i n g on w h e t h e r the
of a different n a t u r e . T h e y t a k e u p t h e m e d i u m micelles are ordered radially or concentrically.
in w h i c h t h e y a r e i m m e r s e d a n d if t h e micelles T h e s e different c a t e g o r i e s a r e d e r i v e d from o n e
are oriented geometrically, with a small spacing a n o t h e r in a c c o r d a n c e w i t h t h e following
c o m p a r e d w i t h t h e w a v e l e n g t h of t h e light u s e d , s c h e m e w h i c h m a k e s it e a s y to r e m e m b e r t h e m :
o n e sees s h a p e b i r e f r i n g e n c e o r s t r u c t u r a l biref- isotropic > foliated > tubular >
r i n g e n c e . If t h e micelles t h e m s e l v e s a r e c r y s t a l - ^ helical 1 ^ annular ^ negative spherulitic
line, t h e i r p r i m a r y b i r e f r i n g e n c e is s u p e r i m - ^ ^ fibrous > positive spherulitic
posed on the structural birefringence, resulting
I n s o m e w a y , t h e fact t h a t t h e micelles a r e
in c o m p l e x b i r e f r i n g e n c e . T h e s e different t y p e s
l a m i n a t e d b e t w e e n t w o l i m i t i n g surfaces l e a d s to
of b i r e f r i n g e n c e a r e s t u d i e d b y i m m e r s i n g t h e
t h e o c c u r r e n c e of i s o t r o p i c a n d foliated s t r u c -
o b j e c t s in l i q u i d s of a c c u r a t e l y k n o w n refractive
tures. W h e n this l a m i n a t i o n takes place on a
i n d i c e s . S t r u c t u r a l b i r e f r i n g e n c e is e l i m i n a t e d
cylinder, a t u b u l a r structure arises. Elongation
w h e n t h e i m m e r s i o n l i q u i d h a s a refractive
of t h i s c y l i n d e r , c o u p l e d w i t h a twist, gives a
i n d e x e q u a l to t h a t of t h e c r y s t a l l i n e m i c e l l e s .
h e l i c a l s t r u c t u r e . A x i a l c o m p r e s s i o n r e s u l t s in
Biological b i r e f r i n g e n c e s a r e classified u n d e r
a n n u l a r s t r u c t u r e , w h i l e a h i g h d e g r e e of e l o n g a -
six c a t e g o r i e s , u s i n g a n o m e n c l a t u r e w h i c h is
t i o n gives a fibrous s t r u c t u r e . R e d u c t i o n of t h e
i n d i c a t i v e of t h e i r i n f r a s t r u c t u r e . I n p a s s i n g , it
c y l i n d e r to a s p h e r e gives a p o s i t i v e o r n e g a t i v e
m a y be mentioned that electron microscopes
s p h e r u l i t i c s t r u c t u r e , d e p e n d i n g o n w h e t h e r it
have completely confirmed the schematic struc-
d e r i v e s from o n e o r t h e o t h e r of t h e l a t t e r t w o
t u r e s set u p o n t h e b a s i s of p o l a r i z a t i o n s t u d i e s ,
structures.
e s p e c i a l l y t h o s e for t h e 'foliated' s t r u c t u r e s fre-
q u e n t l y e n c o u n t e r e d o n t h e surface of s p o r e s COUNTER-PLATED AND MOSAIC STRUCTURES
a n d pollens. T h e above-mentioned categories W h e n s u p e r i m p o s e d l a y e r s of t h e c o n s t i t u e n t s of
a r e t h e following: fibrous; a n n u l a r ; helical; t u b u - a m e m b r a n e are orientated at a constant angle
lar; foliated; s p h e r u l i t i c . r e l a t i v e to e a c h o t h e r , s u c h a s t r u c t u r e is c a l l e d
I n fibrous s t r u c t u r e s t h e micelles a r e o r i e n t e d counter-plated. These have been studied parti-
a l o n g t h e m a j o r axis of t h e o b j e c t w i t h a q u a s i - cularly by Bouligand (1980). W h e n the succes-
crystalline regularity. In the longitudinal direc- sive l a y e r s a r e i d e n t i c a l a n d t h e a n g l e is 90, t h e
t i o n , e x t i n c t i o n is c l e a r , w h i l e in t h e t r a n s v e r s e s t r u c t u r e c a n n o t b e d i s t i n g u i s h e d from a n iso-
d i r e c t i o n t h e r e is a faint o r n o b l a c k cross. t r o p i c s t r u c t u r e w h i c h c a u s e s a superficial effect
Annular structures show the opposite tenden- o n it w h i c h a l t e r s t h e b i r e f r i n g e n c e of o n e of t h e
cies to t h e p r e c e d i n g o n e s : t h e micelles a r e t w o l a y e r s . I n g e n e r a l , t h e s w e l l i n g of a n a n i s o -
o r i e n t e d a t r i g h t a n g l e s to t h e m a j o r a x i s . E x - t r o p i c s t r u c t u r e is a l w a y s a c c o m p a n i e d b y a
t i n c t i o n in t h e l o n g i t u d i n a l d i r e c t i o n is c l e a r , b u t d i m i n u t i o n of t h e b i r e f r i n g e n c e .
n e g a t i v e w i t h r e s p e c t to e x t e n s i o n in a low i n d e x M o s a i c structures are those whose overall
m e d i u m , w h i l e a b l a c k cross is o b s e r v e d in t h e a n i s o t r o p y v a r i e s from o n e side to t h e o t h e r of
transverse direction. t h e s a m e s t r u c t u r e . S u c h s t r u c t u r e s a r e well seen
H e l i c a l s t r u c t u r e s , often referred to i n c o r r e c t - by scanning electron microscopy when there are
ly a s s p i r a l s t r u c t u r e s , d o n o t p r o d u c e c o m p l e t e e n o u g h micelles.
e x t i n c t i o n ; o n t h e o t h e r h a n d , a t r a n s v e r s e sec-
t i o n o r a p a r t i a l l y fibrous m a t e r i a l in t h e l o n g i - METACHROMATIC DICHROISM
tudinal direction displays oblique extinction. W h e n t h e micelles of a n a n i s o t r o p i c s t r u c t u r e
T u b u l a r s t r u c t u r e s a r e helical s t r u c t u r e s w i t h a r e s i t u a t e d w i t h i n a n i s o t r o p i c m a t r i x , in s o m e
foliated t e x t u r e . c a s e s it is p o s s i b l e to differentiate b e t w e e n t h e
F o l i a t e d s t r u c t u r e s s h o w s t r a i g h t e x t i n c t i o n in c o n s t i t u e n t s b y s t u d y i n g t h e i r m e t a c h r o m a s y in
o n e or two directions b u t are statistically isotro- p o l a r i z e d light, if o n e of t h e c o n s t i t u e n t s c a n b e
p i c a n d h e n c e w i t h o u t e x t i n c t i o n in a t h i r d m a d e metachromatic with a metachromatic
d i r e c t i o n w h i c h is a l w a y s p a r a l l e l to t h e surface; s t a i n . L o c q u i n a n d V e n e c - P e y r (1979) h a v e
they are structures oriented by lamination be- d e v e l o p e d a t e c h n i q u e t h e y call ' c h r o m o t r o p i c
Chief instrumental methods 49

dichroism'. T h e specimen stained with a


m e t a c h r o m a t i c b l u e is o b s e r v e d b e t w e e n c r o s s -
polars; the isotropic matrix remains blue a n d
t h e a n i s o t r o p i c m i c e l l e s a p p e a r r e d in a s u i t a b l e
orientation between two cross-polars.
BG 12 PE OG 1

Fluorescence microscopy
F l u o r e s c e n c e m i c r o s c o p y is b a s e d o n t h e
observation that certain substances, when sub-
j e c t e d to i n t e n s e i r r a d i a t i o n , a b s o r b r a d i a t i o n s
of s h o r t w a v e l e n g t h s a n d e m i t r a d i a t i o n s of KG 1 UG 1 BG38 PE GG 13c
l o n g e r w a v e l e n g t h s (Figure 1.53), k n o w n a s
F i g u r e 1.54. Principle of fluorescence microscopy.
Aboveunder blue light; belowunder ultraviolet
light. K G 1, heat-absorbing filter; U G 1, ultraviolet
filter; BG38, blue filter which cuts off red light; PE,
object plane; GG 13c, filter which cuts off ultraviolet
rays; BG 12, blue filter; O G 1, orange filter for stopping
blue

b o t h a s r e g a r d s t h e lenses a n d t h e b a l s a m in
which they are sealed. T h e m o u n t i n g m e d i u m
u s e d for t h e p r e p a r a t i o n a s well a s t h e i m m e r -
s i o n oil m u s t a l s o b e free from f l u o r e s c e n c e . I t is
a d v i s a b l e to c h e c k t h i s b y p r e p a r i n g a b l a n k
Figure 1.53. Quantum principle of fluorescence. An s p e c i m e n , n o t c o n t a i n i n g a n y fluorescent o b -
incident ray of short wavelength () excites an electron in j e c t s . S o m e f l u o r e s c e n t d y e s tuffs a c t e v e n w h e n
the electron cloud surrounding the atomic nucleus at the
centre; the energy absorbed by the electron causes it to be
p r e s e n t in e x t r e m e l y s m a l l a m o u n t s . A b s o l u t e
propelled to the peripheral layers of the cloud from which it cleanliness a n d the greatest possible care m u s t
returns by quantum jumps, each time emitting fluorescence be exercised to avoid these adventitious p h e -
radiation of longer wavelengths ( 2 and then 3) nomena.
(Wild-Heerbrugg)
D i r e c t e x a m i n a t i o n w i t h p h a s e c o n t r a s t is
f l u o r e s c e n c e ; this is t h e S t o k e s effect. T o d e m o n - s o m e t i m e s c o m b i n e d with fluorescence micros-
strate this p h e n o m e n o n , the exciting b a n d c o p y in o r d e r to b e a b l e to localize a c c u r a t e l y
w h i c h is g e n e r a l l y u l t r a v i o l e t o r b l u i s h violet, t h e f l u o r e s c e n t s o u r c e s in cells o r tissues (Figure
m u s t b e clearly d e m a r c a t e d w i t h a first c o l o u r 1.55).
filter. A s e c o n d ' l o w p a s s ' filter is t h e n p l a c e d M e n t i o n m a y b e m a d e h e r e of t h e e x a m i n a -
b e h i n d t h e object. T h i s c u t s off t h e r e m a i n i n g t i o n of f l u o r e s c e n c e r a d i a t i o n m a k i n g u s e of
t r a c e s of t h e e x c i t i n g r a d i a t i o n , o n l y a l l o w i n g polarization, either by polarizing the exciting
t h e f l u o r e s c e n c e to p a s s t h r o u g h . T h e p r o c e s s r a d i a t i o n itself o r b y a n a l y s i n g t h e p o l a r i z a t i o n
m a y b e i m p r o v e d b y m e a n s of v a r i o u s o p t i c a l of t h e f l u o r e s c e n c e r a d i a t i o n o n l y , w i t h t h e a i d of
d e v i c e s . T h e o b j e c t to b e e x c i t e d is i l l u m i n a t e d an analyser. C.A. Baud and subsequently M .
a g a i n s t a b l a c k b a c k g r o u n d in o r d e r to e n s u r e L o c q u i n h a v e s h o w n t h a t it is p o s s i b l e to d e d u c e
t h a t d i r e c t light d o e s n o t e n t e r t h e o b j e c t i v e ; t h e t h i s f r o m t h e o r i e n t a t i o n of t h e m i c e l l e s a l o n g
o p t i m u m b l a c k b a c k g r o u n d for t h i s c a s e is a w h i c h t h e f l u o r o c h r o m e u n d e r g o e s fixation.
c e n t r a l b l a c k b a c k g r o u n d , m a k i n g u s e of a M i c r o f l u o r e s c e n c e is s a i d to b e p r i m a r y if t h e
S p i e r e r - L o c q u i n objective with an o p a q u e cen- o b j e c t s t h e m s e l v e s a r e fluorescent, a n d i n d u c e d
tral lozenge. T h e object can also be illuminated o r s e c o n d a r y if a dyestuff is a t t a c k e d selectively
from a b o v e , t h r o u g h t h e o b j e c t i v e o r u s i n g a n o n a s t r u c t u r e a n d t h e dyestuff fluoresces. I f a
e p i - i l l u m i n a t o r . T h e c a t a l o g u e s of v a r i o u s d e s i g - f l u o r o c h r o m e is a t t a c h e d to a l a b e l l e d a n t i b o d y ,
n e r s c o n t a i n e l e g a n t i l l u s t r a t i o n s of t h e s e dif- t h i s is k n o w n a s i m m u n o f l u o r e s c e n c e , a t e c h n i -
ferent a p p l i a n c e s (Figure 1.54). q u e u s e d in i m m u n o h i s t o c h e m i s t r y .
O b j e c t i v e s u s e d for f l u o r e s c e n t e x a m i n a t i o n s T h e s e a r e t h e t h r e e b a s i c v a r i a n t s of m i c r o -
s h o u l d t h e m s e l v e s b e free from f l u o r e s c e n c e fluorescence.
50 Instruments and techniques

a n t i g e n to b e identified, d i r e c t i m m u n o f l u o r e s c -
ence results; however, one can detect antigens
w h i c h h a v e b e c o m e b o u n d to t h e i r a n t i b o d i e s b y
an indirect method.
T h e s e r o l o g i c a l a s p e c t of t h i s t o p i c is of g r e a t
i m p o r t a n c e , a n d c a n b e f o u n d in s p e c i a l i z e d
t e x t b o o k s . T h e dyestuff a s p e c t w h i c h is also of
c o n s i d e r a b l e i m p o r t a n c e a n d r e l e v a n t to m i c r o s -
c o p y will b e d e a l t w i t h in C h a p t e r 3 ( p . 176).

IMMUNOFLUORESCENCE
K a p l a n a n d K r a f t (1965) u s e d i m m u n o f l u o r e s c -
e n c e to s h o w u p p a t h o g e n i c fungi in h u m a n
t i s s u e s . T h e y d e m o n s t r a t e d t h e cells of t h e fungi
b y d i g e s t i o n w i t h 1 % t r y p s i n , after formol fixa-
t i o n , c u t t i n g , a n d m o u n t i n g o n a slide. T h e
s t a i n i n g is d o n e w i t h fluorescein b o u n d to t h e
a n t i - g l o b u l i n of t h e cells u n d e r s t u d y , s u c h a s
Blastomycetes, Coccidiomycetes or Cryptococci.
T h e s a m e p r i n c i p l e a p p l i e s to e l e c t r o n m i c r o s -
F i g u r e 1.55. Light path in a c o p y in w h i c h l a b e l l i n g m a y l e a d e i t h e r to t r u e
mixed phase electron fluorescence when using a p p a r a t u s
contrast/fluorescence e q u i p p e d to d e t e c t fluorescent e l e c t r o n s , o r to a n
microscope. Interest in this i n t e n s i f i c a t i o n of c o n t r a s t s a s in t h e i m m u n o -
method lies in the possibility
of superimposing on the
ferritin m e t h o d .
fluorescence image an image
having structural details of CATHODE LUMINESCENCE
another colour Different k i n d s of s i g n a l a r e e m i t t e d b y a n
(Leitz-Wetzlar) e l e c t r o n i c s o u r c e , s u c h a s X - r a y s , A u g e r elec-
t r o n s , diffracted e l e c t r o n s , diffused e l e c t r o n s a n d
c a t h o d e l u m i n e s c e n c e . S o m e of t h e s e c o n d a r y
SPECIFIC LABELLING W I T H F L U O R O C H R O M E S
e m i s s i o n s give u s i n f o r m a t i o n a b o u t t h e a t o m i c
T h i s c o n s t i t u t e s t h e b a s i s of i m m u n o f l u o r e s c -
c o n s t i t u e n t s of t h e s u b s t a n c e s t h e y s t r i k e , o t h e r s
e n c e . I m m u n i t y is a defensive m e c h a n i s m a c -
a b o u t the crystal structures, while others require
q u i r e d b y a living o r g a n i s m , w h i l e r e s i s t a n c e is
more complex interpretations.
natural and inherent. W e distinguish between
C a t h o d e l u m i n e s c e n c e is e x c i t e d in low p r e s s -
a c t i v e i m m u n i t y p r o d u c e d b y d i r e c t injection of
u r e b y a b e a m of e l e c t r o n s . A b o v e all, it p e r m i t s
m i l d t o x i n s o r of t h e w e a k e n e d p a t h o g e n itself,
o n e to r e d u c e t h e d a m a g e d o n e to t h e s p e c i m e n
a n d p a s s i v e i m m u n i t y , w h i c h is o b t a i n e d b y
t o a m i n i m u m b y r e d u c i n g its e n e r g y , a n d
i n j e c t i n g a n t i b o d i e s f o r m e d in a n o t h e r a n i m a l .
s c a n n i n g the specimen as rapidly as possible.
I n all t h e s e c a s e s , t h e t e r m a n t i g e n is a p p l i e d to
T h e s e c o n d a r y p h o t o n s p r o d u c e d a t t h e surface
s u b s t a n c e s w h i c h p r o v o k e a defensive r e a c t i o n ,
a r e scattered, a n d this p e r m i t s only the relative-
a n d t h e t e r m a n t i b o d y to s u b s t a n c e s p r o d u c e d
ly p o o r r e s o l u t i o n of . C a t h o d e l u m i n e s c -
to c o m b a t the antigen. I m m u n i t y , w h e t h e r ac-
e n c e , o r p r e f e r a b l y t h e e l e c t r o l u m i n e s c e n c e of
tive o r p a s s i v e , r e s u l t s in a g g l u t i n a t i o n , p r e -
c i p i t a t i o n o r v a r i o u s o t h e r r e a c t i o n s , o n e of L o c q u i n (1981), has a n i m p o r t a n t future as a
which takes place w h e n antigens are labelled t e c h n i q u e in c y t o l o g y a n d h i s t o l o g y for fixed
with fluorochromes. Both antigens a n d anti- cells, a n d for t h e p r e c i s e l o c a l i z a t i o n of n a t u r a l l y
b o d i e s m a y b e l a b e l l e d ; if t h e t w o s u b s t a n c e s fluorescent c o m p o u n d s in living cells. C o n n e c -
c o m b i n e , t h e m o l e c u l e s to w h i c h a fluorescent t i o n of t h e i n s t r u m e n t w i t h a s p e c t r o g r a p h p e r -
d y e s t u f f h a s b e e n a t t a c h e d to l a b e l t h e m b e c o m e m i t s o n e to m a k e a n a c c u r a t e a n a l y s i s of t h e
a t t a c h e d selectively to t h e i r c o m p l e m e n t a n d fluorescent components.
p r o v i d e e v i d e n c e for its p r e s e n c e b y i n t e n s e MICROELECTROLUMINESCENCE
fluorescence w h i c h is e a s y to l o c a t e . W h e n u s i n g I n s t e a d of e x c i t i n g fluorescence b y u l t r a v i o l e t
a s e r u m c o n t a i n i n g specific a n t i b o d i e s for t h e l i g h t , L o c q u i n , in 1 9 8 1 , p r o p o s e d t h e i d e a of
Chief instrumental methods 51

e l e c t r i c a l e x c i t a t i o n . T h e s p e c i m e n is p u t in m a y , m o r e o v e r , b e u s e d a s a n o r m a l c a m e r a to
c o n t a c t w i t h a l i g h t - s e n s i t i v e e m u l s i o n o n a flat t r a n s m i t p i c t u r e s of m a n i p u l a t i o n s o r to t r a n s -
e l e c t r o d e w h i c h c r e a t e s a n e l e c t r i c field. T h e m i t l a r g e i m a g e s of s m a l l o b j e c t s . T h e following
e m u l s i o n is d e v e l o p e d , a n d t h e i m a g e a p p e a r s p o i n t s m u s t b e o b s e r v e d w h e n u s i n g a c a m e r a in
u n d e r the microscope as a n a u t o r a d i o g r a p h , combination with a microscope.
which can be enlarged subsequently. T h u s one T h e cameras should preferably be operated
c a n o b s e r v e in situ t h e fluorescence of t h e living close to t h e h o r i z o n t a l to a v o i d s h o c k s o r v i b r a -
cytoplasm without any staining. tions which might cause minute particles within
t h e s e n s i t i v e t u b e to b e c o m e d e t a c h e d . T h e s e
DOUBLE FLUORESCENCE m a y fall o n t h e s e n s i t i v e surface a n d c a u s e s p o t s
Fluorochromes which induce dichroism may which m a y degrade the image. Nowadays, most
i n d u c e fluorescent d i c h r o i s m . T h e p h e n o m e n o n d e s i g n e r s of m i c r o s c o p e s p r o v i d e s u i t a b l e
c a n b e s e e n w i t h a single p o l a r o c u l a r . T h e mechanical adapters. T h e user should then
d i c h r o i c c o l o u r e d s t r u c t u r e gives o u t p o l a r i z e d select a n a d e q u a t e o p t i c a l s y s t e m . S i n c e t h e
l i g h t w h i c h is d e t e c t e d w h e n it is p a r a l l e l to t h e c a m e r a t u b e g e n e r a l l y h a s a useful size e q u a l to
p l a n e of v i b r a t i o n of t h e p o l a r i z e d o c u l a r . I n t h a t of a 16 m m film c a m e r a , c o m p a r a b l e e n -
o r d e r to m a k e t h e d i c h r o i s m easily visible, o n e l a r g e m e n t s on the sensitive surface should be
u s e s s t a i n s like t h i a z o l e y e l l o w w h i c h gives a u s e d , b e a r i n g in m i n d t h a t t h e definition of a
brilliant fluorescence against the deep blue film is g r e a t l y s u p e r i o r to t h a t of t h e v i d e c o n
w h e n t h e p o l a r filter is a t 90. D o u b l e fluoresc- c u r r e n t l y u s e d w i t h television c a m e r a s . T h e
e n c e , like t h e differential d i s p e r s a l b y diffrac- l a t t e r g e n e r a l l y u s e s a 2 0 lines p e r m i l l i m e t r e
t i o n , d e p e n d s u p o n t h e refractive i n d e x of t h e s y s t e m . T h e r e s o l u t i o n c a n b e c a l c u l a t e d a s lines
m e d i u m in w h i c h t h e s p e c i m e n is e x a m i n e d . p e r m i l l i m e t r e of t h e p r i n c i p a l objectives b y
C l e r i c i r e c o m m e n d s a n a q u e o u s s o l u t i o n of t h a l - d i v i d i n g t h e v a l u e c o r r e s p o n d i n g to t h e o b j e c -
lium formate a n d thallium m a l o n a t e whose re- tive used by 2 0 ; one thus obtains the o p t i m u m
fractive i n d i c e s lie b e t w e e n 1.35 a n d 1.75. e n l a r g e m e n t o n t h e v i d e c o n t u b e d i r e c t l y , for
i n s t a n c e 2 0 0 in t h e c a s e of a n i m m e r s i o n o b j e c -
QUENCHING OF FLUORESCENCE tive w i t h a 1.20 a p e r t u r e a n d 4 0 for a X 1 0
I t is w o r t h n o t i n g t h a t i o d i n e q u e n c h e s t h e o b j e c t i v e h a v i n g a n a p e r t u r e of 0 . 2 5 . I t c a n b e
fluorescence of m o l e c u l e s o u t s i d e n u c l e a r D N A , seen that these enlargements generally corres-
b u t silver q u e n c h e s i n t e r n a l fluorescence. p o n d to one-fifth of t h o s e u s e d in p h o t o m i c -
r o g r a p h y o r w i t h d i r e c t o b s e r v a t i o n . S u c h low
magnifications require weak eyepieces, short
Projection m i c r o s c o p y
extensions a n d a correcting lens. T h e observer
A projection microscope transports the image m u s t b e a t a n o p t i m u m d i s t a n c e from t h e s c r e e n
i s s u i n g from a m i c r o s c o p e t h r o u g h a c e r t a i n w h i c h c o r r e s p o n d s to 7 t i m e s its size in o r d e r to
d i s t a n c e w h i c h m a y r a n g e from s e v e r a l d e c i - c a p t u r e all t h e d e t a i l s of t h e i m a g e o n t h e
m e t r e s to s e v e r a l h u n d r e d m e t r e s . F o r t h i s r e c e i v e r s c r e e n . W h e n h e is t o o close, his vision
p u r p o s e , t h r e e b a s i c t y p e s of e q u i p m e n t a r e is i m p a i r e d b y t h e s c a n n i n g lines of t h e i m a g e ,
used: w h e r e a s if h e is t o o far, h e loses s o m e r e s o l u t i o n
s i n c e t h e eye is u n a b l e to p e r c e i v e d e t a i l s s e p a -
( 1 ) O p t i c a l v e h i c l e s of t h e t y p e f o u n d in
r a t e d b y a n a n g l e of less t h a n 2 ' . T h i s l i m i t a t i o n
p e r i s c o p e s , w h i c h a r e effective m a i n l y w h e n t h e
i m p o s e d b y t h e eye also a p p l i e s to a n i m a g e
m i c r o s c o p e is u s e d in a r a d i o a c t i v e m e d i u m .
p r o j e c t e d d i r e c t l y b y t h e m i c r o s c o p e o n to a
( 2 ) T h e t r a n s p o r t of i m a g e s b y c o h e r e n t a n d
s c r e e n , b u t in t h i s c a s e t h e r e is n o i n t e r m e d i a t e
flexible fibre o p t i c s ; t h e s e a r e g r a d u a l l y r e p l a c -
l i m i t a t i o n a n d , in p r i n c i p l e , e y e p i e c e m a g n i f i c a -
i n g t h e first t y p e of a p p l i a n c e o n a c c o u n t of t h e i r
t i o n is t h e n s e l e c t e d in a c c o r d a n c e w i t h t h e
flexibility, w h i c h m a k e s it p o s s i b l e to s e p a r a t e
desired projection distance.
the receiver by mechanical m e a n s .
(3) C l o s e d c i r c u i t television c a m e r a s in w h i c h By analogy with magnetic tape recorders used
t h e c o n n e c t i o n b e t w e e n e m i t t e r a n d r e c e i v e r is t o r e c o r d s o u n d , t h e r e a r e m a g n t o s c o p e s for
m a d e b y w i r e . T h i s offers t h e a d v a n t a g e t h a t r e c o r d i n g i m a g e s d i r e c t l y from a c a m e r a . T h e s e
m u l t i p l e r e c e i v e r s c a n b e u s e d in o n e r o o m o r in a r e a v a i l a b l e b o t h for b l a c k a n d w h i t e a n d for
different r o o m s , w h i c h is n o t p o s s i b l e w i t h t h e c o l o u r . T h e y h a v e t h r e e m a i n a p p l i c a t i o n s in
first t w o t y p e s of e q u i p m e n t . T h e s a m e c a m e r a m i c r o s c o p y : t h e y m a k e it p o s s i b l e to p r e s e r v e
52 Instruments and techniques

a n d r e - e x a m i n e u n c o m m o n o r fleeting i m a g e s ; of light s o u r c e s , lack of sensitivity of t h e e m u l -


t h e y m a k e it p o s s i b l e to e d i t s e q u e n c e s ; w h e n s i o n s , a n d lack of s t a b i l i t y of t h e e q u i p m e n t .
i n s t a l l e d in p a r a l l e l w i t h t h e film c a m e r a o n a P r o g r e s s m a d e d u r i n g t h e p a s t few y e a r s h a s
c i n e m i c r o g r a p h i c i n s t a l l a t i o n , t h e y m a k e it r e s u l t e d in t h e p r o d u c t i o n of c o m p a c t e q u i p -
p o s s i b l e , before d e v e l o p i n g t h e film, to r e c o r d m e n t w h i c h c a n f u n c t i o n u n d e r all c o n d i t i o n s
t h e final view w h i c h p r o v i d e s a v i s u a l c h e c k o n (Figure 1.56).
t h e film s e q u e n c e .
T e l e v i s i o n t u b e s a r e s u p e r i o r to t h e eye a n d to
films in o n e r e s p e c t ; t h e y a r e h i g h l y sensitive to
w e a k i l l u m i n a t i o n . F o r this r e a s o n t h e o b j e c t
d o e s n o t suffer d a z z l i n g o r o v e r h e a t i n g w h i c h
m i g h t affect its b e h a v i o u r o r c a u s e it to lose its
c o l o u r . A n e l e c t r o n i c s y s t e m r e g u l a t e s t h e level
of light a m p l i f i c a t i o n a u t o m a t i c a l l y a n d this
f r e q u e n t l y m a k e s it p o s s i b l e to r e g u l a t e c o n t r a s t
w i t h t h e a i d of t h e c o n d e n s e r d i a p h r a g m , w i t h -
o u t h a v i n g to r e a d j u s t t h e o v e r a l l level of illu-
mination.
B y w a y of e x a m p l e , it is c u r r e n t l y p o s s i b l e to
v a r y t h e i n t e n s i t y of i l l u m i n a t i o n from 1 to 20
w i t h o u t affecting t h e i m a g e a n d w i t h t h e illu-
m i n a t i o n o n t h e t u b e 1/4000 of t h a t u s e d in
p h o t o m i c r o g r a p h y with the s a m e objective.

W h e n a television t u b e h a s b e e n d a z z l e d b y
excessively s t r o n g light, i r r e g u l a r i t i e s in t h e
sensitivity of its surface a p p e a r as a l t e r n a t i v e
d a r k a n d light tails o n t h e i m a g e . I n s u c h a c a s e ,
o n e m u s t s w i t c h off t h e i l l u m i n a t i o n of t h e
m i c r o s c o p e a n d l e a v e t h e c a m e r a o n for s e v e r a l
m i n u t e s o r h o u r s u n t i l it h a s r e c o v e r e d its initial
s e n s i t i v i t y . F a i l u r e to d o so m a y l e a d to a
c o n s i d e r a b l e s h o r t e n i n g of t h e w o r k i n g life of t h e
tube. A very bright spot s u r r o u n d e d by a d a r k
h a l o o n t h e i m a g e c a n b e m a d e to d i s a p p e a r b y
diminishing the illumination or by adjusting the
photocathode.

F i g u r e 1.56. Diamirex B B T research microscope with


photographic equipment and interference contrast (BBT,
Paris)
1.4 Recording of images
W h a t e v e r the a p p a r a t u s used, the utmost care
m u s t b e t a k e n d u r i n g i n s t a l l a t i o n a n d in s u b s e -
Photomicrography
q u e n t u s e to e l i m i n a t e v i b r a t i o n s as far a s
T h e t e r m p h o t o m i c r o g r a p h y h a s c o m e to d e n o t e possible. A suitable alternative involves m o u n t -
a photograph taken through the microscope, ing the whole assembly on an anti-vibration
w h e r e a s m i c r o p h o t o g r a p h y refers to p h o t o - s u p p o r t w h i c h isolates it from its e n v i r o n m e n t .
g r a p h i c m i c r o c o p i e s of d o c u m e n t s . T h e s e t w o E l i m i n a t i o n of v i b r a t i o n s d u r i n g i n s t a l l a t i o n is
t e r m s a r e f r e q u e n t l y u s e d i n t e r c h a n g e a b l y in t h e e s s e n t i a l l y a m a t t e r of d e s i g n , a l t h o u g h t h e
l i t e r a t u r e . F o r t u n a t e l y , t h e r e is r a r e l y a n y a m b i - s u r r o u n d i n g s also p l a y a n i m p o r t a n t p a r t . If t h e
guity, and the two techniques have m a n y com- l a b o r a t o r y is s i t u a t e d in a l a r g e t o w n o r n e x t to a
m o n features. r o a d , it is p r a c t i c a l l y i m p o s s i b l e for t h e f o u n d a -
For a long time, photomicrography at high t i o n s of t h e b u i l d i n g n o t to v i b r a t e w h e n , for
magnification has h a d three problems: weakness e x a m p l e , lorries o r u n d e r g r o u n d t r a i n s p a s s t h e
Recording of images 53

building. Photomicrographic equipment m a y be M a x i m u m p o s s i b l e c l e a n l i n e s s of t h e o p t i c a l


i s o l a t e d from t h e b u i l d i n g b y m e a n s of a fre- s u r f a c e s , i n c l u d i n g p r e p a r a t i o n s , is e v e n m o r e
q u e n c y filter. T h e b e s t f r e q u e n c y filter is a t a n k e s s e n t i a l h e r e t h a n for v i s u a l o b s e r v a t i o n , a s
filled w i t h fine, d r y s a n d , of 150 m m t h i c k n e s s , well a s t h e g r e a t e s t p o s s i b l e u n i f o r m i t y of illu-
on which rests a heavy, iron plate which does m i n a t i o n . All t h e c e n t r i n g s m u s t b e carefully
n o t t o u c h t h e sides of t h e t a n k a t a n y p o i n t . c h e c k e d in t h e following o r d e r : c e n t r i n g of t h e
D a m p i n g c l o t h s , r u b b e r b l o c k s a n d all o t h e r filament w i t h r e s p e c t to its collector, c e n t r i n g of
d e v i c e s sold a s d a m p i n g d e v i c e s a n d f r e q u e n c y t h e b e a m l e a v i n g t h e l a m p w i t h r e s p e c t to t h e
filters a r e less efficient t h a n a s i m p l e s a n d t a n k . i n l e t iris of t h e c o n d e n s e r , c e n t r i n g of t h e c o n -
S o m e v i b r a t i o n s a r e p r o d u c e d in t h e e q u i p - d e n s e r w i t h r e s p e c t to t h e o b j e c t i v e , c e n t r i n g of
m e n t itself a n d c a n n o t b e c o m p l e t e l y e l i m i n a t e d : t h e field d i a p h r a g m w i t h r e s p e c t to t h e p h o t o -
t h e s e i n c l u d e r a i s i n g of t h e m i r r o r in reflecting g r a p h i c field, c e n t r i n g of t h e i l l u m i n a t i o n r i n g s
a p p a r a t u s e s or shaking w h e n o p e n i n g or closing w i t h r e s p e c t to t h e p h a s e p l a t e , c e n t r i n g of t h e
a focal p l a n e s h u t t e r . F o r t h i s r e a s o n , it is c h a m b e r w i t h r e s p e c t to t h e e y e p i e c e .
advisable with high power magnification always F o c u s i n g m u s t b e m a d e v e r y carefully. F i r s t ,
t o u s e e i t h e r a s h o r t e x p o s u r e of less t h a n 0.02 s t h e c o n d e n s e r m u s t p r o j e c t a s h a r p i m a g e of t h e
o r a l o n g e x p o s u r e of m o r e t h a n 2 s. I n t h e first field d i a p h r a g m o n to t h e p l a n e of t h e o b j e c t to
case, mechanical vibrations which have a longer be p h o t o g r a p h e d ; next, the sighting eyepiece
p e r i o d c a u s e n o t r o u b l e , w h i l e in t h e s e c o n d m u s t b e focused o n to t h e focusing a n d c e n t r i n g
case, they are d a m p e n e d d u r i n g a small initial c r o s s - w i r e s ; finally, t h e o p t i c a l s y s t e m of t h e
fraction of t h e e x p o s u r e t i m e w i t h o u t d a m a g e to m i c r o s c o p e is focused o n to t h e object. P a r a l l a c -
the image. tic f o c u s i n g c a n n o t b e too h i g h l y r e c o m m e n d e d ,
s i n c e t h i s a v o i d s t r o u b l e s o m e a d j u s t m e n t s of t h e
C H O I C E OF E Q U I P M E N T o b s e r v e r ' s eye w h e n following t h e i m a g e . T h e
W h e n choosing the equipment, one should take p r i n c i p l e of t h i s m e t h o d of focusing is a s follows:
i n t o a c c o u n t , first, t h e f r e q u e n c y of t a k i n g if t h e o b j e c t i m a g e is in t h e s a m e p l a n e a s t h e
p h o t o g r a p h s r e l a t i v e to t h e n u m b e r of o b s e r v a - c o r r e c t l y focused v i e w i n g c r o s s - w i r e s , a l a t e r a l
t i o n s a n d , s e c o n d l y , t h e d e g r e e of h o m o g e n e i t y d i s p l a c e m e n t of t h e eye b e h i n d t h e e y e p i e c e d o e s
w h e n t a k i n g p h o t o g r a p h s , in r e s p e c t of t h e n o t p r o d u c e a n y d i s p l a c e m e n t of o n e i m a g e w i t h
v a r i e t y of s u b j e c t s , c o l o u r s a n d m a g n i f i c a t i o n s . r e s p e c t to t h e o t h e r . All t h a t n e e d s to b e d o n e to
I f t h e f r e q u e n c y of t a k i n g p h o t o g r a p h s is low c h e c k t h i s is to m o v e t h e h e a d b a c k w a r d s a n d
compared with the observation time, equipment f o r w a r d s slightly b e h i n d t h e v i e w i n g s y s t e m : if
w h i c h t a k e s a r e l a t i v e l y l o n g t i m e to m a n i p u l a t e f o c u s i n g is c o r r e c t , t h e o b j e c t r e m a i n s fixed w i t h
a n d w h i c h c a n b e p o s i t i o n e d a t t h e m o m e n t of r e s p e c t to t h e c r o s s - w i r e s .
t a k i n g t h e p h o t o g r a p h c a n b e a d a p t e d for u s e . Before u s i n g a p i e c e of e q u i p m e n t o n e s h o u l d
H o w e v e r , if a l a r g e n u m b e r of n e g a t i v e s h a v e to c h e c k t h a t t h e r e is n o e x t r a n e o u s light f o r m i n g a
b e t a k e n d u r i n g t h e c o u r s e of t h e o b s e r v a t i o n s , h a l o o r a c e n t r a l , o v e r - e x p o s e d p o i n t in t h e
the apparatus must be permanently mounted on i m a g e ; after all t h e a d j u s t m e n t s h a v e b e e n c a r -
t h e m i c r o s c o p e , offering t h e p o s s i b i l i t y of p r o - r i e d o u t o n t h e o b j e c t , it c a n b e r e m o v e d a n d t h e
c e e d i n g a l m o s t i n s t a n t a n e o u s l y from o b s e r v a - i n s i d e of t h e t u b e of t h e e q u i p m e n t i n s p e c t e d
t i o n to p h o t o g r a p h y . If p o s s i b l e , t h e e x p o s u r e w i t h t h e e y e p i e c e t a k e n o u t . If s o m e reflections
time should be m a d e automatic. a r e o b s e r v e d o n t h e fixing c o l l a r of t h e r e v o l v i n g
I f t a k i n g of t h e p h o t o g r a p h s c a n b e s t a n d a r - n o s e p i e c e o r o n t h e i n s i d e of t h e t u b e , t h e
d i z e d , i.e. if t h e o b j e c t s a r e r e l a t i v e l y u n i f o r m in a p p a r a t u s h a s to b e a d j u s t e d . O n e s h o u l d also
n a t u r e , c o l o u r a n d m a g n i f i c a t i o n , it is a d v a n - c h e c k t h a t t h e e y e p i e c e lens h a s b e e n a d j u s t e d
t a g e o u s to w o r k t h r o u g h o u t w i t h t h e s a m e p r o p e r l y in t e r m s of its e x t e n s i o n ; if t h e c h a m b e r
s o u r c e , t h e s a m e filter, t h e s a m e e m u l s i o n a n d a h a s a fixed focal e x t e n s i o n , t h i s is a set d e s i g n
c o n s t a n t i l l u m i n a t i o n ; t h e m e a s u r i n g cell u n i t factor; if t h e e x t e n s i o n is v a r i a b l e , a n e y e p i e c e
for t h e e x p o s u r e t i m e t h e n s e r v e s s i m p l y to w i t h a d j u s t a b l e lens m u s t b e u s e d . A l t h o u g h this
e n s u r e i d e n t i c a l c o n d i t i o n s for s u c c e s s i v e o p e r a - e y e p i e c e is often p r o v i d e d w i t h g r a d u a t e d m a r k -
tions. i n g s s h o w i n g t h e e x t e n s i o n , it is still g r e a t l y
T h e different light s o u r c e s a r e i n d i c a t e d in p r e f e r a b l e to m a k i n g a d j u s t m e n t s w h i l e o b s e r v -
S e c t i o n 1.2 ( p . 13) a n d a v a i l a b l e o p t i c a l e q u i p - i n g t h e i m a g e , in w h i c h c i r c u m s t a n c e all t h e
m e n t is s h o w n in Figures 1.57 a n d 1.58. correction factors can be c o m b i n e d ; these are:
54

Figure 1.57. Apophot system of Nikon. This shows the microscope at the centre and all accessories which may be attached together or separately. Where
these are joined by a line this signifies that they are interchangeable but cannot be used together (Nikon, Japan)
Figure 1.58. Multiphot system of Nikon for photomacrography. Same remarks apply as for Figure 1.57 (Nikon, Japan)
55
56 Instruments and techniques

t u b e l e n g t h , e x t e n s i o n of t h e c h a m b e r , t h i c k n e s s is a d v i s a b l e to u s e a s p e c i a l c o n d e n s e r w i t h a
of t h e c o v e r - s l i p , refractive i n d e x a n d t h i c k n e s s l a r g e field a n d to p l a c e a g r o u n d glass p l a t e in
of t h e m o u n t i n g m e d i u m , a n d w a v e l e n g t h of t h e t h e p a t h of t h e b e a m of l i g h t . I f t h e g r o u n d g l a s s
i l l u m i n a t i n g light. is so c o a r s e g r a i n e d t h a t it m a y b e seen s u p e r i m -
posed on the image, the grain m a y be reduced
b y r u b b i n g oil o n t h e g r o u n d s u r f a c e a n d w i p i n g
Photomacrography
off a n y excess w i t h filter p a p e r , u n t i l t h e d e s i r e d
By dfinition, t h i s is p h o t o m i c r o g r a p h y a t m a g - g r a i n t h i c k n e s s h a s b e e n o b t a i n e d . A n oiled
nification o n t h e film b e t w e e n 2 a n d 50. filter r e t a i n s t h e s e c h a r a c t e r i s t i c s for a l o n g t i m e ,
A l t h o u g h p o s s i b l e , it is n o t r e c o m m e n d e d t h a t b u t e v e n t u a l l y t h e oil e v a p o r a t e s a n d t h e g r a i n
t h e m i c r o s c o p e b e u s e d to t a k e p h o t o g r a p h s of reappears gradually.
t h i s t y p e . V e r y low p o w e r objectives t e n d to b e I n p h o t o m a c r o g r a p h y , less s t r o n g l y c o n t r a s t -
c h o s e n a n d , in g e n e r a l , t h e s e d o n o t h a v e a l a r g e i n g e m u l s i o n s a r e u s e d m o r e often t h a n in
e n o u g h field to r e c o r d w h a t is v i e w e d b y t h e eye p h o t o m i c r o g r a p h y . O n e uses the s a m e contrast

F i g u r e 1.59. Illumination of a transparent object in photomacrography. The condenser C contains one or two lenses. It
projects an image of the source on the iris of the objective L through the object Ob. There are no diaphragms

in t h e s t e r e o s c o p i c m i c r o s c o p e . W h e n e v e r t h i s is filters, filters b a l a n c i n g t h e c o l o u r t e m p e r a t u r e s
p o s s i b l e w i t h t h e p a r t i c u l a r object, it is prefer- a n d h e a t - a b s o r b i n g filters. W h e n p h o t o g r a p h s
a b l e to t r a n s f e r it to t h e s p e c i a l i z e d e q u i p m e n t a r e t a k e n of o b j e c t s in w a t e r o r m o u n t e d in a
w h i c h comprises specially interchangeable transparent m e d i u m illuminated by incident
objectives b u t no eyepiece. For this, one either l i g h t , t h e r e is t h e a d v a n t a g e t h a t o n e c a n e l i m i n -
u s e s a specially a d a p t e d o b j e c t i v e o r a n i n v e r t e d a t e reflections w i t h a p o l a r i z e r p l a c e d in t h e
o b j e c t i v e of a s m a l l p h o t o g r a p h i c a p p a r a t u s . o b s e r v a t i o n s y s t e m , in front of t h e v i e w i n g sys-
I n p h o t o m a c r o g r a p h y , o n e often n e e d s to t e m a n d a d j u s t e d to t h e eye.
r e c o r d t h e i m a g e s of o b j e c t s h a v i n g a definite
o u t l i n e , v i e w e d b y reflection. I n this c a s e , it is
m u c h m o r e difficult to a c h i e v e c o r r e c t i l l u m i n a -
Illumination d u r i n g photomicrography
tion because one requires at the s a m e time a
o n e - d i r e c t i o n a l effect b r i n g i n g o u t t h e o u t l i n e , T h e r e a r e a s m a n y w a y s of c a r r y i n g o u t p h o t o -
a n d sufficiently u n i f o r m b a c k g r o u n d i l l u m i n a - m i c r o g r a p h y as t h e r e a r e m e t h o d s of i l l u m i n a -
t i o n to s h o w d e t a i l s clearly. t i o n . T h e r e a d e r s h o u l d refer to t h e r e l e v a n t
W h e n i l l u m i n a t i n g w i t h t r a n s m i t t e d light, it c h a p t e r for a p a r t i c u l a r c a s e .
Recording of images 57

I L L U M I N A T I O N OF O P A Q U E OBJECTS U S I N G (1) I s t h e c o n t r a s t in t h e o b j e c t sufficient?


INCIDENT LIGHT (2) H a s t h e r e s o l u t i o n b e e n c h o s e n c o r r e c t l y ?
I l l u m i n a t i o n c a n b e o b l i q u e , c e n t r a l o r from all (3) I s t h e c o l o u r of t h e i l l u m i n a t i n g light o p t i m -
s i d e s . I n t h e first c a s e , t h e w o r k i n g d i s t a n c e of al?
t h e o b j e c t i v e m u s t b e l a r g e e n o u g h to e n a b l e t h e (4) I s t h e p r e p a r a t i o n satisfactory?
b e a m of l i g h t f r o m t h e l a m p t o b e d i r e c t e d o n t o (5) H a s t h e e m u l s i o n b e e n c h o s e n c o r r e c t l y ?
t h e o b j e c t . I n t h e s e c o n d c a s e , a b e a m of l i g h t is (6) I s b r i g h t field i l l u m i n a t i o n t h e b e s t c h o i c e
d i r e c t e d t h r o u g h t h e o b j e c t i v e o n to t h e o b j e c t (very i m p o r t a n t ) ?
b y a s p e c i a l light s o u r c e . I n t h e t h i r d c a s e , t h e
o b j e c t i v e is s u r r o u n d e d b y a r i n g - s h a p e d sys- C o n t r a s t m u s t b e e x a m i n e d in r e l a t i o n to all t h e
t e m . W h e n t h e i n c i d e n t b e a m of l i g h t p a s s e s o t h e r questions, since contrast d e p e n d s on the
t h r o u g h t h e o b j e c t i v e , a n i m a g e is f o r m e d in a resolution, the colour contrast, the preparation,
b r i g h t field. W h e n t h e i n c i d e n t b e a m of l i g h t is t h e g r a i n a n d t h e c o n t r a s t of t h e e m u l s i o n . W e
o b l i q u e o r from all s i d e s , t h e i m a g e is l o c a t e d in h a v e p u t t h i s p o i n t first, b e c a u s e it e n c o m p a s s e s
a d a r k field. A p p r o p r i a t e s o u r c e s of i l l u m i n a t i o n all t h e o t h e r factors a n d o n e c a n , t h e r e f o r e ,
h a v e d e v e l o p e d g r e a t l y , p a r t i c u l a r l y for s t u d y - decide immediately whether modifications must
i n g m e t a l l i c s u r f a c e s . T h e y c a n , h o w e v e r , also b e t r i e d in o r d e r to i m p r o v e t h e q u a l i t y of t h e
b e u s e d in b i o l o g y . Figure 1.36 s h o w s s c h e m a t i - i m a g e . W h e n a p o o r q u a l i t y i m a g e is f o r m e d ,
cally t h e p a t h t r a v e l l e d b y t h e r a y s of light in t h e o n e c a n n o t d e t e r m i n e w h e t h e r this h a s b e e n
m a i n t y p e s of s y s t e m s w i t h i n c i d e n t light. c a u s e d b y i n a d e q u a t e r e s o l v i n g p o w e r of t h e
objective until one has examined systematically
all p o s s i b l e o t h e r c a u s e s .
DARK G R O U N D ILLUMINATION
T o find o u t w h e t h e r t h e b e s t c o l o u r e d filter
A n i n t e n s e s o u r c e m u s t b e u s e d to o b t a i n
h a s b e e n u s e d , t h e r e a d e r s h o u l d refer to t h e
reasonable exposure times. T h e r e m u s t only be
s e c t i o n b e l o w o n c o l o u r e d filters. I n d e a l i n g w i t h
a s m a l l n u m b e r of o b j e c t s in t h e field a n d t h e s e
a n u n f a m i l i a r o b j e c t for e x a m i n a t i o n o n e d o e s
must never be superimposed. T h e r e must be no
n o t a s k first w h e t h e r t h e p r e p a r a t i o n is satisfac-
a i r b u b b l e s in t h e m o u n t i n g m e d i u m o r in t h e
t o r y , b e c a u s e t h e o p e r a t o r h a s n o p o i n t s of
immersion liquid. At high power magnification,
reference. O n the other h a n d , d u r i n g routine
t h e front lens of t h e c o n d e n s e r m u s t b e in
w o r k o n s t a n d a r d test o b j e c t s , t h i s is o b v i o u s l y
c o n t a c t w i t h t h e p r e p a r a t i o n in t h e s a m e i m m e r -
t h e first q u e s t i o n to b e s e t t l e d .
sion l i q u i d a s t h e o b j e c t i v e , w h i c h c a n c a u s e
F o r t h e b e s t c h o i c e of e m u l s i o n see l a t e r in
p r o b l e m s if t h e o b j e c t is d i s p l a c e d . I n d a r k field
this section (p.62).
i l l u m i n a t i o n , c e n t r i n g of t h e c o n d e n s e r a n d
I n all t h e p r e c e d i n g w o r k , it h a s b e e n
f o c u s i n g o n t h e o b j e c t a r e e x t r e m e l y c r i t i c a l . I t is
a s s u m e d t h a t t h e c h o i c e of b r i g h t field i l l u m i n a -
a l w a y s a d v i s a b l e to c a r r y o u t t h e s e a d j u s t m e n t s
t i o n w a s m a d e w i t h full k n o w l e d g e of all t h e
progressively while increasing the magnifica-
variables.
tion.

B R I G H T FIELD I L L U M I N A T I O N Coloured filters


T h i s is t h e m o s t c o m m o n t y p e of i l l u m i n a t i o n .
F r o m t h e p o i n t of v i e w of t h e i r p h y s i c a l p r o p -
F o r p h o t o g r a p h y , t h e following factors a r e
e r t i e s , t h e r e a r e t h r e e t y p e s of c o l o u r e d filter,
associated with this type: positive p h a s e con- w h i c h a l t e r t h e c h r o m a t i c c o m p o s i t i o n of t h e
trast; interference contrast; images formed by i l l u m i n a t i n g b e a m of light, u s u a l l y w h i t e light,
polarization with compensators. O n the other which passes through them.
h a n d , i m a g e s f o r m e d in d a r k field i l l u m i n a t i o n
are bound u p with negative phase contrast at (1) L o w p a s s filters, w h i c h s t o p light of l o n g
h i g h p o w e r a b s o r p t i o n a s well a s i m a g e s b e - w a v e l e n g t h s , b u t a l l o w l i g h t of s h o r t
t w e e n c r o s s e d niois w i t h o u t c o m p e n s a t o r s , a n d w a v e l e n g t h s to p a s s t h r o u g h .
f l u o r e s c e n c e i m a g e s in w h i c h o n l y few d e t a i l s (2) H i g h p a s s filters, w h i c h s t o p light of s h o r t
are fluorescent. w a v e l e n g t h s , b u t a l l o w l i g h t of l o n g
Photographic negatives are rarely better t h a n w a v e l e n g t h s to p a s s t h r o u g h .
t h e c o r r e s p o n d i n g i m a g e s o b s e r v e d w i t h t h e eye (3) B a n d - p a s s filters, w h i c h a l l o w o n e o r s e v e r a l
a n d it is n e c e s s a r y , t h e r e f o r e , to p o s e s o m e b a n d s of light l y i n g w i t h i n a p a r t i c u l a r
q u e s t i o n s before t a k i n g p h o t o g r a p h s : s p e c t r a l r a n g e to p a s s t h r o u g h (Figure 1.60).
58 Instruments and techniques

(1) H e a t - a b s o r b i n g filters, p r e f e r a b l y interfer-


^" -;;; Pale yellow e n c e , o r ' c o l d m i r r o r ' i n t e r f e r e n c e filters.
R
:, .F : ;. Dark yellow
(2) C o l o u r c o m p e n s a t o r filtersa series of 36
K o d a k - W r a t t e n g e l a t i n filters. T h i s c o m -
Orange p l e t e series of L o c q u i n Chromotaxia filters
m o u n t e d o n n e e d l e s in a fan-tail a r r a n g e -
Magenta
m e n t a l l o w s t h e o p e r a t o r to find t h e b e s t
;.^V:s!- Purple
c o n t r a s t filter q u i c k l y w i t h o u t h a v i n g s p e -
H cial k n o w l e d g e of c o l o u r (cf. p p . 6 1 - 6 2 ) .
' [- T V\o\e\ (3) M o n o c h r o m a t i c filters, p r e f e r a b l y interfer-
e n c e filters, to select o n e line in t h e s p e c -
Light blue
t r u m of a d i s c h a r g e l a m p o r to set o u t a
X 400 500 600 700 nm b r o a d line w i t h a c o n t i n u o u s s p e c t r a l s o u r c e
Blue Green Red (cf. t h e r e l e v a n t t a b l e for t h e c h o i c e of lines
Figure 1.60. Position of the absorption bands and to select).
colours transmitted by the filters (4) C o m p l e m e n t a r y h i g h p a s s a n d low p a s s
filters for f l u o r e s c e n c e ; t h e low p a s s filter
T h e c o m b i n a t i o n of a low p a s s filter w i t h a s e r v e s a s a n e x c i t a t i o n filter, t h e h i g h p a s s
h i g h p a s s filter b y s u p e r p o s i t i o n c a n r e s u l t in filter is u s e d for o b s e r v a t i o n (cf. p p . 4 9 - 5 1 ) .
t h e f o r m a t i o n of a b a n d - p a s s filter if t h e r e g i o n (5) D i d y m u s p o l y c h r o m a t i c filters to intensify
transmitted by one overlaps the region transmit- t h e c o l o u r c o n t r a s t s . T h i s filter is w i d e l y
t e d b y t h e o t h e r . A h e a t - a b s o r b i n g filter is a u s e d for o b s e r v a t i o n a n d p h o t o g r a p h y . T h e
b a n d - p a s s filter for all visible r a y s . m o s t useful c o n t r a s t filters a r e c h o s e n
B a n d - p a s s filters c o n s i s t of g e l a t i n . ( K o d a k - a c c o r d i n g to w h e t h e r o n e w i s h e s to o b t a i n
W r a t t e n filters) o r g l a s s , o r t h e y m a y b e i n t e r - o p t i m u m definition w i t h o n e c o l o u r o r o p t i -
f e r e n c e , c o m p o s i t e , l i q u i d o r p o l a r i z i n g filters. m u m c o n t r a s t w i t h r e g a r d to t h e r a n g e of
I n t e r f e r e n c e filters h a v e t h e a d v a n t a g e o v e r t h e c o l o u r s p r e s e n t in t h e o b j e c t . U s u a l l y t h e s e
o t h e r s of h a v i n g a p a s s b a n d w i t h s h a r p p e a k s t w o a i m s a r e i n c o m p a t i b l e , especially in
a n d t h e y a l l o w a slight d i s p l a c e m e n t of t h e p a s s black and white.
b a n d to b e effected b y r o t a t i o n a b o u t a n axis
MONOCHROMATIC PHOTOGRAPHY
p e r p e n d i c u l a r to t h e b e a m of light. O n t h e o t h e r
W h e n p h o t o g r a p h y is c a r r i e d o u t to o b t a i n a
h a n d , they are generally more absorbent.
n e g a t i v e w i t h t h e finest p o s s i b l e d e t a i l s of a
O n e r e q u i r e s t h e following filters to o b s e r v e c o l o u r l e s s o r m o n o c h r o m a t i c object, o n e s h o u l d
different c o l o u r e d o b j e c t s : u s e a m o n o c h r o m a t i c filter of t h e s h o r t e s t p o s s i -

T a b l e 1.1 Kodak-Wratten colour correction filter taken from Chromotaxia*

Maximum Yellow (Y) Coefficient Magenta (M) Coefficient Cyano (C) Coefficient
density (absorbs blue) (absorbs green) (absorbs red)

05 CC-05 Y CC-05 M 1.3 CC-05 C 1.3


10 CC-10Y 1.3 CC-10M 1.3 CC-10C 1.3
20 CC-20 Y 1.3 CC-20 M 1.3 CC-20 C 1.3
30 CC-30Y 1.3 CC-30 M 1.6 CC-30 C 1.6
40 CC-40Y 1.3 CC-40 M 1.6 CC-40 C 1.6
50 CC-50 Y 1.6 CC-50 M 1.6 CC-50 C 2.0

Maximum Red (R) Coefficient Green (G) Coefficient Blue (B) Coefficient
density (absorbs blue (absorbs blue (absorbs red
and green) and red) and green)

05 CC-05 R 1.3 CC-05 G 1.3 CC-05 1.3


10 CC-10R 1.3 CC-10G 1.3 CC-10B 1.3
20 CC-20 R 1.3 CC-20 G 1.3 CC-20 1.6
30 CC-30 R 1.6 CC-30 G 1.6 CC-30 1.6
40 CC-40 R 1.6 CC-40 G 1.6 CC-40 2.0
50 CC-50 R 2.0 CC-50 G 2.0 CC-50 2.3
*The range of colours and densities of these different filters makes it possible to apply useful corrections in most cases.
Recording of images 59


80 {
+ o
i /
1

1 \
* f1 s
60 y< \ / i 1
1

1
\ *1 .:
/ 1
J2
/
3 i 4

5 l: 6 7
/
40 / j! 1 f
+


1
\
/:
/ ;
1
1 +

M
\
20| / \\ /* j 1
il 0+

\

\ \

/
! \ ; : 1;
\ \ : \X
M\

J y
- -t F i g u r e 1.61. Series of 7 Balzers
V
350 400 450 500 550 600

%T

50
Red
90
s
/ *-\
1I
^ 525nm
80 > y 40
fr fi- y
I 1 \
\
I 1I \
I i 1
60 1 \ 30
rI G reen
I j 1 >
/

I
/
/ 12.5 > n m
40 j 20
1 /
1 l
1 l
20 1 1 10
1
1 i
;
\j
/
/
Blue
-J V 3 2 r im

400 500 600 700 800 900 nm 400 500 600 700 nm

Figure 1.62. Lefta series of three D T filters; rightFiltraflex monochromatic interference filter (Balzers, Liechtenstein)

b l e w a v e l e n g t h , c o m p a t i b l e w i t h o b j e c t i v e cor- p r a e s o d y m i u m ) glass filters. T h e s e filters h a v e


r e c t i o n s a n d t h e c o l o u r of t h e o b j e c t (Figures 1.61 multiple absorption b a n d s which are fortunately
a n d 1.62). l o c a t e d a t t h e b o u n d a r y of t h e m a j o r r e g i o n of
s e n s i t i v i t y of t h e e y e to c o l o u r . T h e y i n c r e a s e t h e
POLYCHROMATIC COLOUR PHOTOGRAPHY c l a r i t y of t h e c o l o u r s a n d so c a n p a r t i a l l y o v e r -
If t h e o b j e c t h a s s e v e r a l c o l o u r s a n d m u s t b e c o m e t h e g r a d u a l loss of c l a r i t y p r o d u c e d b y t h e
p h o t o g r a p h e d in c o l o u r , t h e q u a l i t y of t h e r e p r o - c o l o u r e m u l s i o n s . Table .II (see A p p e n d i x I)
d u c t i o n of t h e c o l o u r e m u l s i o n s c a n often b e gives t h e f o r m u l a of a l i q u i d n e o d y m i u m filter.
d e c e p t i v e if t h e b a n d t r a n s m i t t e d b y t h e s t a i n I n t e r f e r e n c e p o l y c h r o m a t i c filters c a n a l s o b e
u s e d lies, for e x a m p l e , b e t w e e n t w o of t h e t h r e e u s e d in c o m b i n a t i o n w i t h d i d y m i u m filters,
p r i m a r y c o l o u r s of t h e e m u l s i o n . I n t h i s c a s e , w h i c h offer t h e a d v a n t a g e t h a t t h e p o s i t i o n of
w h i c h u n f o r t u n a t e l y o c c u r s f r e q u e n t l y , it is r e - the absorption b a n d s can be varied by inclining
c o m m e n d e d that one should use liquid neo- t h e filter r e l a t i v e to t h e b e a m of light. T h e m o s t
d y m i u m filters of d i d y m i u m (neodymium- useful p o l y c h r o m a t i c i n t e r f e r e n c e filters a r e
60 Instruments and techniques

t h o s e w i t h t h r e e o r five b a n d s . N e v e r t h e l e s s , r a p i d e v a p o r a t i o n of t h e g a s , p r o d u c i n g a b l a c k -
t h e y suffer from o n e d r a w b a c k , i.e. t h e p o s i t i o n e n i n g of t h e b u l b a n d h e n c e a d r o p in t h e
a n d d i s p l a y of t h e b a n d s is n o t a s g o o d as w i t h luminosity a n d the colour temperature. A great
d i d y m i u m filters. d e a l of p r o g r e s s h a s b e e n m a d e w i t h q u a r t z -
R h e i n b e r g ' s differential c o l o u r e d i l l u m i n a - iodine a n d latterly with q u a r t z - h a l o g e n bulbs.
t i o n s c a n also b e u s e d in t h r e e different w a y s : I n t h e s e , t h e a d d i t i o n of i o d i n e o r a m i x t u r e of
b r o m i n e a n d i o d i n e to t h e k r y p t o n o r x e n o n in
(1) C o l o u r e d filters c u t i n t o t w o t i m e s t w o
the b u l b allows at the s a m e time a considerable
q u a d r a n t s , differently c o l o u r e d a n d p l a c e d
r e d u c t i o n in t h e v o l u m e of t h e g a s a n d a r a i s e d
in t h e filter c a r r i e r of t h e c o n d e n s e r . A
t e m p e r a t u r e , b e c a u s e t h e t u n g s t e n m o l e c u l e s of
definite a z i m u t h a l effect is o b t a i n e d b y
t h e filament, w h i c h e v a p o r a t e s as a r e s u l t of
rotating the assembly.
heating, c o m b i n e with the halogens at high
(2) C o n c e n t r i c filters w i t h a b r i g h t field c o n -
t e m p e r a t u r e s a n d become redeposited on the
d e n s e r . I n t h i s c a s e , it is b e s t to p l a c e t h e
filament a s a r e s u l t of d i s s o c i a t i o n , t h u s r e g e n e r -
filter of t h e s h o r t e s t w a v e l e n g t h a t t h e
a t i n g it. T h e q u a r t z u s e d for t h e b u l b e n v e l o p e
periphery.
t u r n s b r i g h t r e d d u r i n g t h e o p e r a t i o n . I t is
(3) I n t h e c a s e of filters w i t h c o n d e n s e r s h a v i n g r e s i s t a n t to t h e r m a l s h o c k s a n d r e m a i n s i m -
a m i x e d b r i g h t a n d d a r k field, t h e d a r k field p e r m e a b l e to the gas at high temperatures.
condenser should have the shortest
T h e eye is sensitive to a v a r i a t i o n of 50 a t
w a v e l e n g t h . T h i s is t h e s o l u t i o n w h i c h gives
a b o u t 3 2 0 0 K , b u t d o e s n o t d e t e c t v a r i a t i o n s of
the most detailed and most contrasted im-
less t h a n 2 0 0 to 5 9 0 0 K . E v e r y c o l o u r e m u l -
a g e s . H o w e v e r , it is e s s e n t i a l to i m m e r s e t h e
s i o n is p r o d u c e d for a n o p t i m u m c o l o u r t e m p e r -
front of t h e c o n d e n s e r for h i g h m a g n i f i c a -
a t u r e specified b y t h e m a n u f a c t u r e r . T y p e s of
tion.
g l a s s u s e d for m a n u f a c t u r i n g t h e o p t i c a l s y s t e m
of a m i c r o s c o p e from t h e collector of t h e l a m p to
Colour differences in e m i s s i o n and
reception the eyepiece, including the condenser, the objec-
tive, t h e p r i s m s a n d t h e l a y e r s e p a r a t o r s , a l s o
I n m i c r o s c o p y , t h r e e factors n e a r l y a l w a y s i n t e r -
i n t r o d u c e a c e r t a i n v a r i a t i o n in t h e c o l o u r
fere w i t h t h e o b s e r v a t i o n o r r e c o r d i n g of i m a g e s ,
t e m p e r a t u r e of t h e s o u r c e w h i c h m e a n s t h a t
falsifying t h e final c o l o u r r e p r o d u c t i o n of t h e
even the most a d e q u a t e theoretical precautions
p r o c e s s : t h e c h r o m a t i c e q u i l i b r i u m of t h e i l l u m i -
c a n n e v e r relieve t h e u s e r f r o m t h e a n x i e t y of
n a t i n g s o u r c e , t h e v a r i a b l e c o l o u r sensitivity of
t r y i n g to o b t a i n g o o d c o l o u r p h o t o g r a p h s a n d
t h e eye w i t h r e g a r d s to t h e i l l u m i n a t i o n , a n d t h e
from t r y i n g to e s t a b l i s h for h i m s e l f t h e b e s t
s e n s i t i v i t y of t h e sensitive l a y e r . T h e c h r o m a t i c
o p e r a t i n g c o n d i t i o n s w i t h t h e a i d of c o l o u r
a b e r r a t i o n of t h e object a n d of t h e c o l o u r e d
c o m p e n s a t i n g filters.
filters a r e d e a l t w i t h in t h e p r e v i o u s s e c t i o n .
W h e n t h e s o u r c e is n o t c o n t i n u o u s b u t h a s a
If t h e light s o u r c e u s e d h a s a c o n t i n u o u s
line s p e c t r u m o r is m i x e d w i t h a line s p e c t r u m
s p e c t r u m , w i t h o u t s u p e r i m p o s e d lines, a p h y s i c -
s u p e r i m p o s e d o n a c o n t i n u o u s b a c k g r o u n d , it is
al q u a n t i t y called t h e ' c o l o u r t e m p e r a t u r e ' c a n
i m p o s s i b l e to u s e t h e c o n c e p t of c o l o u r t e m p e r a -
b e defined, w h i c h e x p r e s s e s in s o m e w a y d e v i a -
ture. In general, such sources must not be used
t i o n from perfect w h i t e light; this d e v i a t i o n v e e r s
t o t a k e c o l o u r p h o t o g r a p h s e x c e p t in specific
t o w a r d s yellow a n d t h e n o r a n g e if t h e t e m p e r a -
c a s e s of w h i c h m e n t i o n will b e m a d e . If t h e
t u r e falls b e l o w 5 5 0 0 K , a n d t o w a r d s b l u e if it
c o r r e c t i o n s a r e n o t m a d e b y m e a n s of t h e col-
rises a b o v e this t e m p e r a t u r e . T h i s reference
o u r e d filters, t h e i m a g e r e c o r d e d will b e v e r y
v a l u e h a s b e e n c h o s e n b e c a u s e it is t h e t e m p e r a -
different from t h e i m a g e o b s e r v e d .
t u r e of a l u m i n o u s c l o u d y b u t b r i g h t sky a t a n
e l e v a t i o n of 25 a b o v e t h e h o r i z o n a t n o o n in
F e b r u a r y a n d in N o v e m b e r , a n d a t 7.00 h o r
Black and white photography
17.00 h s u n in J u n e - J u l y in o u r l a t i t u d e s .
I n t h e filaments of electric b u l b s h e a t e d b y t h e O n e e i t h e r a i m s to o b t a i n a g o o d v i e w of t h e
J o u l e effect, t h e c o l o u r t e m p e r a t u r e v a r i e s w i t h w h o l e o b j e c t w i t h t h e full scale of g r e y s a c h i e v -
t h e g a s in t h e b u l b ; a r g o n , k r y p t o n a n d x e n o n i n g g o o d r e p r o d u c t i o n of all c o l o u r n u a n c e s of
e a c h r a i s e t h e c o l o u r t e m p e r a t u r e to a slight t h e o b j e c t , o r to b e a b l e to p u t t o g e t h e r all
e x t e n t in t h i s o r d e r , since t h e y a l l o w a g r e a t e r s i g n i f i c a n t d e t a i l s a n d to b r i n g t h e m o u t in d a r k
rise in t h e t e m p e r a t u r e of t h e filament, w i t h o u t g r e y o r d e e p b l a c k . T h i s r e s u l t is a c h i e v e d in
Recording of images 61

each case by choosing a good colour c o m p e n s a t - t o t h a t for w h i c h t h e e m u l s i o n w a s i n t e n d e d .


i n g filter. T h e u s u a l p r o c e d u r e is a s follows: T h e illumination must be constant and stable,
b o t h in i n t e n s i t y a n d c o l o u r t e m p e r a t u r e . T h e
(1) O n e m a r k s o n e o r t w o c o l o u r s w i t h o r
light source m u s t have a stabilized voltage or a
w i t h o u t c o l o u r e d b a c k g r o u n d s ; for e x a m p l e ,
m i l l i a m m e t e r w h i c h is sufficiently a c c u r a t e to
a n object with red a n d blue details on a
use the s a m e c u r r e n t d u r i n g successive opera-
g r e e n b a c k g r o u n d . I n o r d e r to r e p r e s e n t t h e
t i o n s . I n o r d e r to a d j u s t t h e l i g h t i n t e n s i t y , a n d
r e d s b y m e a n s of d a r k g r e y , it s h o u l d b e
t h e r e f o r e t h e e x p o s u r e t i m e , o n e r e q u i r e s diffus-
p o s s i b l e to u s e a p a n c h r o m a t i c e m u l s i o n
i n g filters for l o w p o w e r objectives e i t h e r of
k n o w n to b e s e n s i t i v e to r e d , a n d p r o v i d e a
W r a t t e n filters o r of t h e K o d a k n e u t r a l N D
c o l o u r c o m p e n s a t o r filter w h i c h s t o p s t h e
s e r i e s . A series of slides o r p a p e r p r i n t s a r e
p a s s a g e of r e d p r e s e n t in t h e w h i t e light of
t a k e n , w i t h o u t p l a c i n g a n y filters in t h e p a t h of
t h e i l l u m i n a t i n g s o u r c e . H o w e v e r , in o r d e r
t h e l i g h t s o u r c e of t h e m i c r o s c o p e , a n d t h e y a r e
to m a k e the black colours a p p e a r as
e x a m i n e d with an intense but non-fluorescent
m e d i u m g r e y in t h e w h o l e s y s t e m , a g a i n s t a
w h i t e l i g h t s o u r c e i t is v e r y i m p o r t a n t n o t to
w h i t e b a c k g r o u n d , it is c o n v e n i e n t to a b s o r b
u s e f l u o r e s c e n t t u b e s , b e c a u s e t h e y e m i t a line
t h e b l u e c o l o u r s slightly, i n c r e a s i n g t h e i r
s p e c t r u m w h i c h b e c o m e s s u p e r i m p o s e d o n to
c o n t r a s t r e l a t i v e to g r e e n . W i t h o u t a g o o d
t h e c o n t i n u o u s s p e c t r u m of t h e i r o u t e r c a s i n g .
k n o w l e d g e of c o l o u r , this m e t h o d m a y
O n e decides on the correct colour compensator
a p p e a r to b e t o o t h e o r e t i c a l a n d it is prefer-
filter, w h i c h r e s t o r e s a w h i t e b a c k g r o u n d to t h e
a b l e to u s e t h e s e c o n d m e t h o d .
p a r t s w h e r e t h e r e is n o object.
(2) T h e 36 filters of t h e K o d a k - W r a t t e n c o l o u r
T e s t p r e p a r a t i o n s c a n also b e p r o d u c e d b y
c o m p e n s a t o r series m u s t b e a v a i l a b l e . I t is
c u t t i n g four s m a l l s q u a r e s from W r a t t e n c o l o u r
c o n v e n i e n t to u s e t h i s c o m p l e t e series
c o m p e n s a t o r g e l a t i n filters a n d m o u n t i n g t h e m
m o u n t e d o n to n e e d l e s , w h i c h c a n b e s p r e a d
b e t w e e n a slide a n d c o v e r - s l i p in t h e s a m e w a y
o u t like a fan ( M . L o c q u i n ' s Chromotaxia,
a s for a n o b j e c t , a n d p h o t o g r a p h i n g t h e m w i t h a
P a r i s ed., A u t u m n 1956). T h e fan is s p r e a d
l o w p o w e r o b j e c t i v e s u p e r i m p o s e d o n a n object.
o u t so t h a t t h e filters a r e a d j a c e n t to e a c h
H o w e v e r , this m e t h o d is l o n g e r a n d less c o m -
o t h e r a n d they are i n t r o d u c e d successively
m o n l y u s e d b e c a u s e it e x c l u d e s s u p e r i m p o s i n g
i n t o t h e p a t h of t h e b e a m of l i g h t . T h e eye
filters. O n t h e o t h e r h a n d , filters c a n easily b e
o b s e r v e s t h e i m a g e a n d a n y m o d i f i c a t i o n s in
s u p e r i m p o s e d o n slides.
c o n t r a s t . W h e n t h e t w o b e s t filters for col-
our and density have been decided on, they
a r e s u p e r i m p o s e d o n e a c h o t h e r to d e t e r - Balanced colour compensation
m i n e w h e t h e r t h e y a r e c o m p a t i b l e , i.e. to
I n o r d e r to o b t a i n g o o d c o l o u r r e p r o d u c t i o n o n a
ensure that they are not complementary
colour emulsion, a satisfactory b a l a n c e m u s t be
w h i c h w o u l d r e s u l t in t h e i n t r o d u c t i o n of
m a i n t a i n e d b e t w e e n t h e c o l o u r t e m p e r a t u r e of
g r e y . I n this p a r t i c u l a r c a s e , it is n e c e s s a r y
t h e s o u r c e , after t h e l i g h t h a s p a s s e d t h r o u g h t h e
t o p r o c e e d b y m e a n s of s u c c e s s i v e a p p r o x -
w h o l e o p t i c a l s y s t e m of t h e m i c r o s c o p e , a n d t h e
i m a t i o n s w i t h slight c h r o m a t i c c h a n g e s of
c o l o u r t e m p e r a t u r e for w h i c h t h e film w a s d e -
o n e of t h e filters b y r e p l a c i n g , for e x a m p l e , a
signed.
r e d filter R . 4 0 w i t h a n o r a n g e o n e o b t a i n e d
T h e r e a r e four c o m m o n l y u s e d m e t h o d s
by s u p e r i m p o s i n g Y.20 yellow a n d R.20 red
t o e n s u r e t h i s . F i l t e r s o n l y assist in m a i n t a i n -
w h i c h gives o r a n g e 4 0 . W i t h a m e d i o c r e
ing a b a l a n c e between yellow a n d blue or
p r e p a r a t i o n , it is often w o r t h w h i l e b e g i n -
m a g e n t a a n d green, because the green-yellow
n i n g a g a i n r a t h e r t h a n a t t e m p t i n g to p r o -
c e n t r a l p o r t i o n of t h e s p e c t r u m is t a k e n as
d u c e g o o d n e g a t i v e from it. T h e factors for
reference.
e x t e n d i n g t h e e x p o s u r e t i m e for t h e 36
W r a t t e n filters for Chromotaxia a r e g i v e n in
t h e 'Coefficient' c o l u m n s of Table 1.1. (1) C o m p e n s a t i o n : excess u l t r a v i o l e t - b l u e . If
t h e n e g a t i v e h a s b e e n a c t e d o n excessively b y
l i g h t of a s h o r t w a v e l e n g t h , a d o m i n a n t b l u e
Procedures for colour photography colour appears. This can be corrected by using a
T h e c o l o u r t e m p e r a t u r e of t h e light w h i c h h a s y e l l o w filter w h i c h c a n m a k e t h e b l u e d i s a p p e a r
passed through the microscope must be adjusted o n t h e slide. T h i s is t h e n i n s e r t e d i n t o t h e
62 Instruments and techniques

microscope illuminating system when taking Variations and deficiencies in colour


p h o t o g r a p h s . T h e filters of t h e K o d a k - W r a t t e n emulsions
series, listed in L o c q u i n ' s Chromotaxia, c a n a l s o
C o l o u r e m u l s i o n s a r e g e n e r a l l y f o r m e d from
b e u s e d . A n a q u e o u s s o l u t i o n of s o d i u m n i t r a t e
three sandwiched layers, deposited on a plastic
w h i c h h a s b e e n d i l u t e d to give t h e d e s i r e d
s u p p o r t a n d p r o t e c t e d b y i n t e r m e d i a t e l a y e r s . If
c o m p e n s a t i o n value m a k e s a n excellent liquid
d u r i n g m a n u f a c t u r e t h e t h i c k n e s s of o n e of t h e
filter. A 1 0 % s o l u t i o n is u s e d as t h e s t a r t i n g
layers varies, even by a small a m o u n t , a n i m b a -
liquid.
l a n c e of c o l o u r s r e s u l t s . T h i s c a n b e rectified b y
(2) C o m p e n s a t i o n for a lack of b l u e . I f t h e r e
a c o l o u r c o m p e n s a t o r filter w i t h a d e n s i t y of
is n o t e n o u g h b l u e w h e n t a k i n g t h e p h o t o g r a p h ,
0 . 1 0 . W h e n c o l o u r films a r e u s e d in l a r g e n u m -
a d o m i n a n t , almost golden yellow colour
b e r s , it is of a d v a n t a g e to b u y a c o n s i g n m e n t
a p p e a r s . T h i s c a n b e rectified w i t h a b l u e o r
bearing the same batch number. W h e n the
g r e y - b l u e filter; t h e l a t t e r is u s u a l l y sufficient.
n u m b e r s a r e different, t h e c o l o u r b a l a n c e h a s to
T h e y also f o r m p a r t of t h e W r a t t e n series; t h e s e
be re-examined.
a r e t h e a n d C filters. A m m o n i a c a l c o p p e r
s u l p h a t e solution makes a n excellent blue liquid I t is v e r y i m p o r t a n t to k n o w w h e t h e r t h e
filter. e x p o s u r e t i m e s h a v e a n y influence o n t h e c o l o u r
equilibrium. Currently, emulsions are produced
COMPENSATION: MAGENTA-GREEN t o g i v e a n e q u i l i b r i u m a t a b o u t 0 . 0 2 - 0 . 0 1 s. If
COMPLEMENT the exposure times are appreciably longer,
O n c e both the above compensations are correct- w h i c h is u s u a l l y t h e c a s e in p h o t o m i c r o g r a p h y ,
ly c a r r i e d o u t , a r e s i d u a l m a g e n t a - g r e e n i m b a - t h e fact t h a t t h e s e n s i t i v i t y of t h e e m u l s i o n
l a n c e m a y a p p e a r . T h i s c a n b e rectified w i t h a decreases with weaker illumination and that the
m a g e n t a filter to r e m o v e t h e d o m i n a n t g r e e n , o r t h i c k n e s s e s of t h e t h r e e l a y e r s a r e n o t i d e n t i c a l ,
vice v e r s a w h i c h is m u c h r a r e r . A n e o d y m i u m causes a colour imbalance.
l i q u i d filter is a l s o v e r y s u i t a b l e . F i l m s k e p t t o o l o n g in h u m i d c o n d i t i o n s o r in
excessive t e m p e r a t u r e s m a y also s h o w a loss of
(3) C o m p e n s a t i o n b y a d j u s t i n g t h e s u p p l y of
colour equilibrium. T h e manufacturers' recom-
t h e s o u r c e . T h i s m u s t o n l y b e u s e d for s m a l l
m e n d a t i o n s s h o u l d b e followed.
c o m p e n s a t i o n s o r for c o m p l e t i n g a n a d j u s t m e n t
I t is n o t a d v i s a b l e to stock films in a r o o m
c a r r i e d o u t b y o n e of t h e o t h e r t w o m e t h o d s .
c o n t a i n i n g c h e m i c a l s s u c h a s f o r m a l d e h y d e , nit-
Generally speaking, modern optical systems
ric acid or a n y volatile substances. T h i s m a y
produce a d o m i n a n t green colour which can be
r e s u l t in c l o u d i n e s s , c o l o u r i m b a l a n c e o r in
rectified w i t h a m a g e n t a o r d i d y m i u m filter.
a l t e r a t i o n s in s e n s i t i v i t y .
(4) E s t i m a t i o n b y d e c a m i r e d v a l u e s . T o esti-
T h e c l o u d i n e s s m e n t i o n e d c a n also b e f o r m e d
m a t e t h e usefulness of a filter in effecting c o m -
a s a r e s u l t of a n u n t i m e l y e x p o s u r e to t h e light o r
p e n s a t i o n , t h e c o l o u r t e m p e r a t u r e s of t h e s o u r c e
s o m e t i m e s by radiations such as X - r a y s n o w
m u s t b e c o n v e r t e d to d e c a m i r e d u n i t s :
u s e d to d e t e c t m e t a l l i c o b j e c t s a t a i r p o r t s .
T h e r e f o r e , o n e is n o t r e c o m m e n d e d to s e n d
1 decamired =
kelvin v a l u a b l e films b y a i r to b e d e v e l o p e d o r to c a r r y
t h e m w h e n flying.
T h e d e c a m i r e d v a l u e of t h e filter to b e u s e d is
T h e c o l o u r t e m p e r a t u r e of t h e e x a m i n i n g
t h e n c a l c u l a t e d b y m e a n s of t h e f o r m u l a
a p p a r a t u s or projector m a y cause a greater or
lesser d e v i a t i o n t o w a r d s t h e y e l l o w e n d of t h e
D e c a film D e c a s o u r c e = D e c a filter
spectrum. A colour equilibrium should never be
e x a m i n e d b y t h e l i g h t from f l u o r e s c e n t t u b e s ;
A 30 W , 5 A s o u r c e o p e r a t i n g a t 2 9 0 0 h a s 34.5
t h e g r e e n m e r c u r y r a y is often p r e s e n t b e c a u s e it
d e c a m i r e d . A n e g a t i v e c o l o u r film h a s , for e x a m -
easily p a s s e s t h r o u g h t h e t u b e c o v e r i n g a n d
p l e , 31.2 d e c a m i r e d . T o rectify it, a 3.2 d e c a -
a d d s a n excess of g r e e n to t h e i m a g e .
m i r e d filter is i n t e r p o s e d . G . B e t t o n (1969) h a s
published a table showing the decamired values
of t h e p r i n c i p a l s o u r c e s a n d p h o t o g r a p h i c e m u l -
Emulsions
sions. Manufacturers should always indicate the
d e c a m i r e d v a l u e s of t h e i r c o l o u r e m u l s i o n s o n T h e c h o i c e of e m u l s i o n m u s t t a k e i n t o c o n s i d -
the packet. e r a t i o n t h e final r e s u l t , w h i c h is n o t a l w a y s t h e
Recording of images 63

n e g a t i v e itself o r t h e p r i n t . I t is often a n i l l u s t r a - c a l c u l a t e d for e a c h m a g n i f i c a t i o n b y u s i n g a


t i o n p r i n t e d in a p u b l i c a t i o n . I t is a l w a y s n e c e s - s e r i e s of e x p o s u r e t i m e s , i n c r e a s i n g b y V2 a t a
s a r y to k e e p a n a c c o u n t of all t h e links in t h e time.
c h a i n , b e c a u s e t h e final r e p r o d u c t i o n d e p e n d s
b o t h o n t h e c r i t i c a l difficulties a n d o n t h e c o m - Measurement of the e x p o s u r e time
p o u n d i n g of defects a n d e r r o r s , s i n c e it is ex-
t r e m e l y r a r e t h a t t w o defects c a n c e l e a c h o t h e r W h a t e v e r t h e s y s t e m u s e d to m e a s u r e t h e illu-
out! m i n a t i o n of a s e n s i t i v e s u r f a c e , in v i e w of t h e
T h e following factors define a n e m u l s i o n : g r e a t d i v e r s i t y of o b j e c t s a n d m e t h o d s of
spectral sensitivity; contrast or resolving p o w e r observation, one must decide whether one
a n d g r a i n ; s p e e d o r s e n s i t i v i t y ; r a n g e of e x p o - w i s h e s to m e a s u r e t h e w h o l e of a n o b j e c t o r
s u r e t i m e s . T h e s e factors a r e i n t e r r e l a t e d a n d p o i n t s of it. A logical w a y of m a k i n g this difficult
e m u l s i o n s a r e n o t o n t h e m a r k e t w h i c h , for choice m u s t be found, a n d one m a y not have
instance, have high resolving power a n d great s p a c e t o u s e all p o s s i b l e v a r i a t i o n s .
speed, or great contrast a n d a n extended spec-
tral range. M E A S U R E M E N T BY I N T E G R A T I O N O F T H E
W H O L E FIELD
Objects u n d e r a microscope generally show
T h e o b j e c t o b s e r v e d c o m b i n e s w i t h its b a c k -
less c o n t r a s t t h a n o b j e c t s s e e n in t h e i r e n v i r o n -
g r o u n d , g i v i n g a n o v e r a l l effect of all t h e l i g h t
m e n t a n d , therefore, high contrast emulsions
c o m i n g f r o m t h e w h o l e field. U n l e s s t h e o b j e c t is
m u s t b e c h o s e n . F o r t u n a t e l y , this c o n t r a s t is
highly o p a q u e , good results can be obtained
usually coupled with a high resolving power a n d
w i t h b r i g h t field i l l u m i n a t i o n w h e n t h e c a l i b r a -
a v e r y fine g r a i n . O n t h e o t h e r h a n d , h o w e v e r ,
t i o n h a s b e e n c a r r i e d o u t c o r r e c t l y , b e c a u s e few
t h e s p e e d is l o w a n d t h e r a n g e of e x p o s u r e t i m e
o b j e c t s , e v e n w h e n c o v e r i n g t h e w h o l e of t h e
is m o d e r a t e .
field of v i s i o n , a b s o r b m o r e t h a n 20% of t h e
i n c i d e n t light. O n t h e o t h e r h a n d , t h e m e t h o d is
Exposure time definitely n o t r e c o m m e n d e d for d a r k g r o u n d
illumination, fluorescence o r p o l a r i z a t i o n , in
T h e o p t i m a l u t i l i z a t i o n of a n e m u l s i o n d e p e n d s
w h i c h t h e fields a r e b l a c k o r d a r k g r e y . I n
mainly u p o n which emulsion one chooses, which
p r a c t i c e , t h i s i n t e g r a t i o n t e n d s to b e p r o p o r t i o n -
is g o v e r n e d b y t h e o b j e c t b e i n g e x a m i n e d a n d
al t o t h e n u m b e r of o b j e c t s in t h e field o r to t h e i r
t h e five factors m e n t i o n e d a b o v e . A l s o , o n e m u s t
surface area, since the b a c k g r o u n d does not
c h o o s e t h e c o r r e c t e x p o s u r e t i m e to e n s u r e o p t i -
affect t h e r e s u l t s . F o r p h a s e c o n t r a s t , interfer-
m u m r e s u l t s for a g i v e n e m u l s i o n . M o s t m o d e r n
ence contrast, polarization with sensitive stains
i n s t r u m e n t s h a v e lightmeters which are sensi-
o r q u a r t e r - w a v e s , t h e m e t h o d is e m i n e n t l y suit-
tive e n o u g h for all w o r k i n g c o n d i t i o n s . O n e
a b l e , e x c e p t in t h e s p e c i a l c a s e of n e g a t i v e
m u s t a l s o k n o w t h e c o r r e c t s p e e d of t h e e m u l -
contrast with strong absorption appearing on a
sion u s e d . T h e s e a r e u s u a l l y e x p r e s s e d a s A S A
dark background.
o r D I N n u m b e r s a n d o n e c a n d o a series of tests
to d e t e r m i n e w h e t h e r the m a n u f a c t u r e r s ' rating LOCALIZED MEASUREMENT
is c o r r e c t . If n o r a t i n g s a r e g i v e n , t h e y c a n b e I f o n e l i m i t s t h e a r e a of m e a s u r e m e n t to a circle
or a square having approximately one-tenth the
Reaction
Temperature d i a m e t e r of t h e field, t h e m e a s u r e m e n t s c a n b e
C l i m i t e d to o n e o b j e c t o r t o a d e t a i l of t h e o b j e c t ,
1 -36
p r o v i d e d t h a t t h e m i c r o s c o p e s t a g e is m o v e d to
2 33 b r i n g it i n t o t h e c e n t r e of t h e field w h e r e t h e
3 -30 m e a s u r e m e n t is g e n e r a l l y m a d e . T h i s h a s t h e
5 -27
s a m e limitations as the previous method.
H o w e v e r , it c a n b e a p p l i e d , for e x a m p l e , to o n e
10 - 2 4
F i g u r e 1.63. Mean corrections specific o b j e c t of i n t e r e s t a n d of g r e a t l y differing
15 - 2 1 to be applied to periods relating d e n s i t y t h a n its s u r r o u n d i n g s .
to chemical reactions such as
20- - 1 8
fixation, staining and
POINT MEASUREMENTS
25 - 1 5 development of
photo-emulsions. Leftthe T h i s is t h e m o s t t i m e c o n s u m i n g a n d t h e m o s t
30 -12 e x p e n s i v e m e t h o d , b u t it is t h e o n l y o n e t h a t
temperature coefficients
64 Instruments and techniques

a l l o w s t h e l u m i n o s i t y a n d c o n t r a s t to b e m e a - Classification
sured at the s a m e time. I n this case the instru-
T h e b e s t p h o t o g r a p h is w o r t h l e s s if it c a n n o t
m e n t a c t s a s a m i c r o p h o t o m e t e r (cf. p . 8 4 ) .
refer b a c k first to t h e n e g a t i v e , t h e n to t h e
T h e r e f o r e , in p r a c t i c e , o v e r a l l m e a s u r e m e n t s a r e
o b j e c t , t h e n to its p r e p a r a t i o n a n d finally to t h e
taken by integration, introducing a correction
o r i g i n a l . A u n i f o r m m e t h o d of classification
factor as a f u n c t i o n of t h e o b j e c t s , d e t e r m i n e d b y
m u s t b e a d o p t e d b e g i n n i n g w i t h a reference
experience or previously worked out by the
n u m b e r for t h e n e g a t i v e . T h i s n u m b e r m u s t b e
designer a n d displayed on the instrument.
carefully d i s p l a y e d o n all p r i n t s a n d slides a n d a
chronological record book should be kept which
Development c o n t a i n s t h e n u m b e r s of p r e p a r a t i o n s a n d / o r
o b j e c t s , t h e i r o r i g i n , m e t h o d of p r e p a r a t i o n ,
T h e c o n t r a s t , g r a i n a n d sensitivity of a n e m u l -
staining a n d m o u n t i n g , the date they were taken
s i o n a r e l a r g e l y g o v e r n e d b y t h e c h o i c e of d e -
a n d t h e n a m e of t h e p h o t o g r a p h e r .
v e l o p e r , t i m e a n d t e m p e r a t u r e . I n all t h e s e
cases, the variables should be standardized.
M a n u f a c t u r e r s of e m u l s i o n s a n d d e v e l o p e r s
Transposition of contrasts
g e n e r a l l y give d e t a i l e d i n s t r u c t i o n s for u s e , r e -
f e r r i n g to t h e e m u l s i o n s w i t h w h i c h t h e y c a n b e At the photographic emulsion stage, various
u s e d to o b t a i n a g i v e n v a l u e of . I n g e n e r a l , t r a n s p o s i t i o n s in c o n t r a s t m a y b e u s e d . H i s t o r -
c o n t r a s t is i n c r e a s e d b y e x t e n d i n g t h e d e v e l o p - ically, t h e first r e c o r d e d t r a n s p o s i t i o n from a
i n g t i m e a n d b y i n c r e a s i n g t h e a l k a l i n i t y of t h e b l a c k a n d w h i t e c o n t r a s t to c o l o u r e d c o n t r a s t in
bath. m i c r o s c o p y b y m e a n s of a p u r e l y p h o t o g r a p h i c
After c o m p l e t i o n of t h e d e v e l o p m e n t , t h e p r o c e s s m u s t b e a c c r e d i t e d to L o c q u i n ( 1 9 5 0 ) .
emulsion can with advantage be dipped into a T h i s p r o c e s s h a s l o n g b e e n r e p l a c e d b y elec-
fixative b a t h of 7 % a c e t i c a c i d . F i x a t i o n is t r o n i c p r o c e d u r e s , b u t m o s t l y a p p l i e d to X - r a y
c a r r i e d o u t l o n g e n o u g h for all visible t r a c e s of n e g a t i v e s , a l t h o u g h it c a n also b e u s e d for
t h e b r o m i d e in t h e e m u l s i o n to d i s a p p e a r . W h e n micrographie negatives.
t h e e m u l s i o n in t h e b a t h is fresh, t h i s c a n o c c u r I t is a l s o p o s s i b l e to s t a r t from a c o l o u r i m a g e
v e r y q u i c k l y , b u t it t a k e s l o n g e r after i n c r e a s e d a n d r e c o r d it o n s p e c i a l c o l o u r e m u l s i o n s w h i c h
u s e of t h e e m u l s i o n ; it s h o u l d b e r e p l a c e d w h e n a r e s e n s i t i v e to t h e n e a r i n f r a r e d o r n e a r u l t r a -
t h e initial t i m e h a s i n c r e a s e d four-fold. W a s h i n g violet r e g i o n of t h e s p e c t r u m a n d t h e n t r a n s p o s e
t a k e s l o n g e r if t h e n e g a t i v e s a r e to b e k e p t for t h e s e b a n d s to t h e visible s p e c t r u m . I n a d d i t i o n ,
a n y l e n g t h of t i m e . If p o l a r o i d n e g a t i v e s a r e to if a c o l o u r television c a m e r a is a v a i l a b l e to t a k e
b e s t o r e d for as l o n g a s p r i n t s , t h e y m u s t b e a s e q u e n c e of p h o t o g r a p h s , t h e t r a n s p o s i t i o n
carefully w a s h e d before g l a z i n g . A w e t t i n g a g e n t c a n b e effected b y i n v e r t i n g t w o o r t h r e e of t h e
s h o u l d b e a d d e d to t h e final r i n s i n g s o l u t i o n three channels. T h e s e colour transpositions are
before d r y i n g . T h i s h e l p s to a v o i d d e p o s i t i o n of n o t simple, b u t they can be m a d e very clear.
t r a c e s of c a l c i u m salts d u r i n g e v a p o r a t i o n of t h e T h e objects studied most frequently u n d e r the
l a s t few d r o p s of w a t e r . D r y i n g s h o u l d b e c a r r i e d microscope are stained histologically. S o m e s u b -
o u t in d u s t - f r e e c o n d i t i o n s . s t a n c e s in tissues o n l y h a v e a n affinity for s p e c i -
fic s t a i n s w h i c h d o n o t a l w a y s p o s s e s s a t r a n s -
m i s s i o n r a n g e c o r r e s p o n d i n g to t h e m a x i m u m
Printing o n paper
p o w e r of differentiation of t h e eye. G o o d t r a n s -
M i c r o s c o p y l a b o r a t o r i e s w a n t i n g to o b t a i n position re-establishes the o p t i m u m conditions
slides o n b e s t - q u a l i t y p a p e r , p r i n t a n d e n l a r g e of c l a r i t y for t h e p r e p a r a t i o n .
t h e i r o w n n e g a t i v e s . T h e y a r e a b l e to o b t a i n t h e D i c h r o m a t i c o r p o l y c h r o m a t i c i n t e r f e r e n c e fil-
b e s t r e s u l t s from t h e i r n e g a t i v e s , in v i e w of t h e i r ters, with 3, 4 or 5 pass b a n d s , allow the
k n o w l e d g e of t h e o b j e c t s . d i s p l a c e m e n t of t h e p a s s b a n d s w i t h t h e a n g l e of
G l o s s o r m a t t w h i t e p a p e r is u s e d ; m a t t p a p e r i n c i d e n c e of t h e light. T h e filters c a n b e
a l l o w s o n e to d o a little t o u c h i n g u p w i t h p e n c i l m o u n t e d o n a r o t a t i n g s u p p o r t w i t h its axis
or with gouache. Gloss p a p e r shows u p d e e p p e r p e n d i c u l a r to t h e b e a m of light; a t little cost
black m u c h better. Paper with calendered grain t h e y c a n b r i n g a b o u t v e r y useful a n d s o m e t i m e s
m u s t n o t b e u s e d e x c e p t for d i s p l a y s . P l a s t i c s p e c t a c u l a r t r a n s p o s i t i o n s a n d c h a n g e s in c o n -
s u p p o r t s a r e also u n u s a b l e . trast.
Recording of images 65
Special effects in photomicrography t h e L o c q u i n t e c h n i q u e for c o l o u r t r a n s p o s i t i o n
EFFECTS P R O D U C E D W H E N T A K I N G (1950). T h e procedures are long and compli-
PHOTOGRAPHS cated, b u t the results are very impressive.
Rheinberg's polychromatic illumination can be
m a d e of a d i s c of t w o c o n c e n t r i c c o l o u r filters EFFECTS A T T H E P R I N T I N G S T A G E H A R D
PRINTING
placed u n d e r the condenser. W h e n refracting
o b j e c t s s u c h a s fibres o r d i a t o m s a r e o b s e r v e d , T h i s is m a d e e i t h e r s t a r t i n g from a n e g a t i v e o r
p u r e l y o p t i c a l c o l o u r effects a r e o b t a i n e d w h i c h f r o m a p o s i t i v e p r i n t of a n o u t l i n e film. P r o v i d e d
a r e e a s y to r e c o r d o n c o l o u r e m u l s i o n s . A n t h e o b j e c t is s u i t a b l e for t h i s t e c h n i q u e , n e g a -
i n t e r e s t i n g v a r i a t i o n c o n s i s t s of c o m b i n i n g t h e tives c a n b e r e p r o d u c e d , w i t h o u t h a v i n g t o p r i n t
t w o c o l o u r s w i t h t w o different m e t h o d s of t h e m . T h i s r e p r e s e n t s a l a r g e s a v i n g in p r i n t i n g
o b s e r v a t i o n , e.g. d a r k field a n d b r i g h t field c o s t s , b u t t h e n e g a t i v e itself m u s t h a v e m u c h
o b s e r v a t i o n o r b r i g h t field a n d n e g a t i v e p h a s e c o n t r a s t a n d no half-tones which the investiga-
contrast. t o r w i s h e s to e x a m i n e .

SUPERIMPOSED POSITIVES A N D NEGATIVES


EFFECTS A T T H E D E V E L O P M E N T STAGE
WITH DISPLACEMENT
D e v e l o p m e n t c a n b e d o n e e i t h e r of t h e o r i g i n a l
T h e s e a r e o b t a i n e d b y slightly d i s p l a c e d s u p e r -
n e g a t i v e o r , m o r e safely, of a n e g a t i v e p r i n t
i m p o s i t i o n of a n e g a t i v e a n d a p o s i t i v e o b t a i n e d
o b t a i n e d from a n i n t e r m e d i a t e p o s i t i v e p r i n t . I n
f r o m t h e s a m e n e g a t i v e , a n d p r i n t i n g a n e w test
t h i s c a s e , t h e r e is n o risk of d e s t r o y i n g t h e
p r i n t from t h i s c o m p o s i t e n e g a t i v e . T h e r e s u l t s
o r i g i n a l if t h e r e s u l t is n o t s a t i s f a c t o r y . O n e
o b t a i n e d s h o w a s t r a n g e r e s e m b l a n c e to t h e
d e v e l o p s t h e n e g a t i v e in o n e - t h i r d of t h e n o r m a l
i m a g e f o r m e d w i t h a differential i n t e r f e r e n c e
t i m e b y e x p o s i n g it to a 4 0 W b u l b a t a d i s t a n c e
c o n t r a s t . T h e r e is a n o b l i q u e s h a d o w effect
of 1 m for 5 s o r m o r e ; o n e t h e n c o n t i n u e s
w h i c h m a k e s t h e o b j e c t a p p e a r in relief. P o s i t i v e
d e v e l o p i n g it d u r i n g t h e o t h e r t w o - t h i r d s of t h e
- n e g a t i v e r e f e r e n c e m a r k i n g is m a d e e a s i e r b y
t o t a l t i m e . I t is fixed a n d r i n s e d , a n d a p r i n t
t r a c i n g t w o d i a g o n a l l y o p p o s e d r e f e r e n c e crosses
m a d e o n h a r d p a p e r . T h i s r e s u l t s in a n ' e d g i n g '
outside the negative.
effect of t h e c o n t o u r s of e q u a l d e n s i t y of t h e
object, p r o d u c i n g a n i m a g e resembling a n out- ISOHELIA
line d r a w i n g .
T h i s is a t y p e of t o n e s e p a r a t i o n w h i c h a l l o w s
certain dense areas to be emphasized a n d others
TONING
t o b e m i n i m i z e d . T h e p r o c e s s s t a r t s from a n
F o r d i r e c t e x a m i n a t i o n , slides w i t h less c o n t r a s t
i n t e r m e d i a t e p o s i t i v e w h i c h is t h e n u s e d to p r i n t
a r e u s e d b e c a u s e t o n i n g a l w a y s intensifies c o n -
negatives on a very h a r d emulsion with increas-
t r a s t . S e p i a t i n t s a r e o b t a i n e d b y first b l e a c h i n g
ing exposure times. T h e three on which the
in a b a t h c o n t a i n i n g 10 g of p o t a s s i u m ferri-
a r e a s of d e n s i t y to b e e m p h a s i z e d a r e s h o w n
c y a n i d e s o l u t i o n , 1 0 - 2 5 g of p o t a s s i u m b r o m i d e
m o s t clearly are chosen. T h e y are copied a n d
a n d 100 m l of d i s t i l l e d w a t e r . T h e n t h e slide is
m a r k e d o n t h e s a m e p o s i t i v e w i t h soft t o n e s .
i m m e r s e d in a b a t h c o n s i s t i n g of a s o l u t i o n of
T h i s is a n a l o g o u s to c o n t r a s t t r a n s p o s i t i o n w i t h
s o d i u m m o n o s u l p h i d e . A h i g h c o n c e n t r a t i o n of
reduced colour.
b r o m i d e d e e p e n s the tone. Blue tints are
o b t a i n e d b y s p o n t a n e o u s l y m i x i n g t h e following SUPERIMPOSITION
t w o s o l u t i o n s : A , 0.2 g of p o t a s s i u m f e r r i c y a n i d e , W h e n n u m b e r i n g o r m a r k i n g , it is s o m e t i m e s
0.4 m l of s u l p h u r i c a c i d , 100 m l of w a t e r ; B , 0.2 g useful t o s u p e r i m p o s e a g r i d o r r e f e r e n c e p o i n -
of a m m o n i a c a l i r o n c i t r a t e , 0.4 m l of s u l p h u r i c t e r s o n to t h e o b j e c t i m a g e w h e n m a k i n g a p r i n t .
a c i d , a n d 100 m l of w a t e r . T h e p a p e r s a r e I f a p o s i t i v e g r i d is s u p e r i m p o s e d o n to t h e
t r e a t e d w i t h t h e a b o v e s o l u t i o n s , after fixation p a p e r , a n e g a t i v e i m a g e of t h i s is o b t a i n e d . I n
a n d careful w a s h i n g . T h e y a r e w a s h e d after o r d e r to p r o d u c e a positive image, two opera-
t o n i n g , b u t too p r o l o n g e d w a s h i n g w e a k e n s t h e tions m u s t be carried out using a negative grid.
tint. T h e s e c o n d e x p o s u r e is m a d e after p l a c i n g t h e
n e g a t i v e in t h e e n l a r g e r .
T R A N S P O S I T I O N OF C O L O U R C O N T R A S T
DENSITIES DEGREE OF COMPLEMENTATION
R e a d i n g of b l a c k a n d w h i t e o b j e c t i m a g e s c o m - I f a n e g a t i v e is s u p e r i m p o s e d o n a p o s i t i v e of t h e
p r i s i n g a l a r g e r a n g e of c o n t r a s t s is f a c i l i t a t e d b y s a m e object, a n almost uniform grey colour
66 Instruments and techniques

r e s u l t s . T h i s m e t h o d w a s u s e d b y L o c q u i n to slices of t h e o b j e c t , w i t h i n t h e focal p l a n e of t h e
design an automatic apparatus, the Ecctron, o b j e c t i v e , w h i c h is o n l y a few t e n t h s of a m i -
for r e c o g n i t i o n of s h a p e s . c r o m e t r e in t h e c a s e of i m m e r s i o n objectives.
A few r u l e s m a y b e followed to o b t a i n m i c r o -
g r a p h s w h i c h r e c o r d a s m u c h as p o s s i b l e of t h e
Equidensitometry
i n f o r m a t i o n o b t a i n e d b y t h e eye. F i r s t of all, t h e
T h e d a r k e n i n g c u r v e of a p h o t o g r a p h i c e m u l - s m a l l e s t n u m e r i c a l a p e r t u r e w h i c h will s h o w u p
sion w i t h a b l a c k a r e a a n d a w h i t e a r e a in a n t h e d e t a i l s of t h e o b j e c t is c h o s e n ; t h e o b j e c t i v e is
o b j e c t s h o w s a classical S - c u r v e . T h i s defines t h e also selected a n d the p h o t o g r a p h s are taken
h a r d n e s s of t h e e m u l s i o n . By s u p e r i m p o s i n g t h i s b e a r i n g in m i n d t h e m a g n i f i c a t i o n o n t h e film
n e g a t i v e i m a g e o n a p o s i t i v e e m u l s i o n , a c u r v e is a n d a l l o w i n g for t h e e n l a r g e m e n t of t h e p r i n t s
o b t a i n e d w h i c h also h a s a n S - s h a p e , b u t its b y n/2 t i m e s t h e final e n l a r g e m e n t o n t h e posi-
s l o p e is i n v e r t e d c o m p a r e d w i t h t h e p r e v i o u s tive p r i n t , after e n l a r g i n g t i m e s .
o n e . F i n e lines w h i c h b r i n g o u t t h e e q u a l d e n s i t y I f t h e d e p t h of t h e o b j e c t is still g r e a t e r t h a n
c o n t o u r s in t h e o b j e c t c a n b e o b t a i n e d b y s u p e r - t h e d e p t h of field of t h e o b j e c t i v e , t h e p h o t o -
imposing the two curves a n d printing them on a g r a p h is t a k e n b y d i v i d i n g t h e e x p o s u r e t i m e
contrasted emulsion. These procedures can be i n t o p e q u a l fractions a n d m o v i n g t h e focus
r e p e a t e d to o b t a i n a first, s e c o n d a n d e v e n t h i r d b e t w e e n e a c h s u c c e s s i v e s h o t so t h a t it c o v e r s
d e r i v a t i v e of t h e i m a g e . t h e w h o l e r a n g e of t h i c k n e s s of t h e object. W h e n
I t h a s b e e n k n o w n for a l o n g t i m e t h a t t h i s o n e d o e s t h i s , t h e focusing s h o u l d n o t b e a d -
two-stage process can be replaced by one opera- j u s t e d c o n t i n u o u s l y b u t in d i s c r e t e s t e p s , so t h a t
tion using a single emulsion by exposing the e a c h p a r t i a l i m a g e is well defined t h r o u g h o u t
s e n s i t i v e s u r f a c e d u r i n g t h e c o u r s e of t h e d e - t h e e x p o s u r e t i m e a l l o w e d to it. T h e differences
v e l o p m e n t (cf. p . 6 5 ) . T h i s is k n o w n as t h e in focus will b e g o v e r n e d b y t h e s t r u c t u r e of t h e
S a b a t t i e r effect. o b j e c t ( r a t h e r t h a n a r i t h m e t i c a l l y ) , to c o n c e n -
E q u i d e n s i t o m e t r y n e c e s s i t a t e s t h e u s e of h a r d trate on i m p o r t a n t features.
emulsions a n d high contrast developers. As this E x p o s u r e t i m e s will b e l o n g , of t h e o r d e r of
t r e a t m e n t of t h e i m a g e b r i n g s o u t n o t o n l y t h e t e n t h s of a s e c o n d to s e v e r a l s e c o n d s ; t h e n u m -
i n h e r e n t c o n t r a s t s of t h e o b j e c t b u t also t h o s e b e r of d i s c r e t e s t e p s will u s u a l l y b e from 2 to 4 ,
which have been introduced by extraneous b u t m a y well b e h i g h e r for o b j e c t s of v e r y h i g h
s o u r c e s , e.g. c o v e r - s l i p s , d i r t , o b j e c t s u p e r i m -
p o s i t i o n , e t c . , it is a d v i s a b l e to p r o c e e d w i t h
great caution.
E q u i d e n s i t o m e t r y m a k e s it p o s s i b l e to p r o -
d u c e curves by photographic methods, without
special equipment. These curves can be
obtained by optical electronic m e t h o d s at a
m u c h h i g h e r cost, b u t t h e y offer a m u c h g r e a t e r
c e r t a i n t y of t h e i n t e r p r e t a t i o n of t h e i m a g e ,
b e c a u s e of t h e possibility of e l i m i n a t i n g ex-
t r a n e o u s b a c k g r o u n d (light o r m a t e r i a l , cf. p .
90). T h u s , an electronic equidensitograph can
b e d e s i g n e d . T h i s t y p e of i n s t r u m e n t n o w p l a y s
a n i n t e g r a l p a r t in q u a n t i m e t r i c m i c r o s c o p e s
w h i c h a r e d e a l t w i t h in S e c t i o n 1.9 ( p . 9 0 ) .

1.5 Synthesis
A m i c r o s c o p i s t c o n s t a n t l y a l t e r s his focus a n d h e F i g u r e 1.64. Photosynthesis produced by superimposition
a c c o m m o d a t e s reflexly to m a k e a c o r t i c a l s y n t h - by Vialane's technique. Chitinomycete, a nanofossil from
the lower palaeozoic (Ancyrochitina). Magnification X300,
esis of t h e o b j e c t w h i c h h e is e x a m i n i n g . T h e r e - three exposures at three different focal lengths, providing a
fore, h e is often d i s a p p o i n t e d b y his p h o t o m i c r o - clear view of the body of the phialosporangia and its coronal
g r a p h s , s i n c e t h e s e a r e o n l y r e c o r d s of v e r y t h i n processes (M. Locquin)
Synthesis 67

p r e f e r t h i s t e c h n i q u e to a n y o t h e r . If t h e o b j e c t is
a s p h e r e of s m a l l d i m e n s i o n s , e.g. a s p o r e o r a
p o l l e n g r a i n , o n e s h o u l d a l w a y s focus o n t h e
e q u a t o r i a l p l a n e a n d t h e p l a n e t a n g e n t i a l to t h e
u p p e r p o l e of t h e s p h e r e . T h i s is a n e x c e l l e n t
m e t h o d w h e n the objects are semi-transparent.
I t c a n n o t b e a p p l i e d to o p a q u e o b j e c t s , a n d is
less c o n v e n i e n t for u s e w i t h a b i n o c u l a r e y e p i e c e
at low magnification.

Stereoscopic photomicrographs
T h e s e can also be p r o d u c e d with a m o v a b l e
c a m e r a alternately positioned at the two observ-
i n g t u b e s , e i t h e r w i t h a single o b j e c t i v e m i c r o -
scope a n d a stage with a universal joint or with a
t h e o d o l i t e w h i c h e n a b l e s t h e o b s e r v e r to r o t a t e
t h e o b j e c t a r o u n d t h e s a g i t t a l axis of t h e i n s t r u -
m e n t between the two exposures; alternatively,
a s p e c i a l d o u b l e c a m e r a m a y b e u s e d (Figures
Figure 1.65. Photosynthesis produced by superimposition 1.66 a n d 1.67).
of two exposures on thin film, taken at two different focal
lengths, one in the plane of the equator and the other at the
PHOTOMICROGRAPHY OF ISOLATED AND
pole of the object. This provides an impression of the
S P H E R I C A L OBJECTS
spherical relief of spores of 12 diameter and renders the
surface details visible. Immersion objective, numerical A s p o r e o r p o l l e n g r a i n in a m i c r o s c o p e field is
aperture 1.30 (M. Locquin) n o t a l w a y s in t h e o p t i m a l p o s i t i o n to b e p h o t o -
g r a p h e d . A careful w o r k e r m a y c o m p r e s s t h e
c o v e r - s l i p lightly to roll t h e s p e c i m e n i n t o a
b e t t e r p o s i t i o n , b u t w h e n t h e p r e s s u r e is r e -
m o v e d , it often goes b a c k to its p r e v i o u s p o s i -
t i o n . F l e n l e y m a n a g e d to fix s p o r e s in p o s i t i o n
l o n g e n o u g h to p h o t o g r a p h t h e m b y u s i n g a
m i c r o m a n i p u l a t o r to p r e s s o n t h e c o v e r - s l i p ; t h e
m o v e m e n t of t h e i n s t r u m e n t m u s t b e v e r y p r e -
cise, a n d s o m e t i m e s o n e h a s to c h a n g e t h e p o i n t
a t w h i c h p r e s s u r e is a p p l i e d to t h e c o v e r - s l i p .

Graphic reconstructions
I t c a n b e v e r y useful to r e c o n s t r u c t a n o b j e c t in
s u c h a m a n n e r t h a t it c a n b e r e a d in d e p t h like a
geographical m a p with contours. This can be
achieved by using Lison's technique; drawings
of c o n t o u r s a r e m a d e of i m p o r t a n t f e a t u r e s of t h e
o b j e c t from a series of paraffin s e c t i o n s , a n d t h e y
F i g u r e 1.66. Wild's stereoscopic microscope, with
o r t h e i r t r a c i n g s a r e s t a c k e d o n a single b o a r d .
automatic photographic equipment (Wild-Heerbrugg)
A n i n t e r e s t i n g v a r i a n t is to r e - p l o t e a c h d r a w i n g
c o n t r a s t . Figures 1.64 a n d 1.65 i l l u s t r a t e t h e a s if it w e r e b e i n g v i e w e d from a n a n g l e of, s a y ,
excellent results which m a y be achieved by the 45, to o b t a i n a g e n e r a l v i e w of t h e a s s e m b l y
m e t h o d with three-dimensional objects. after s t a c k i n g .
W i t h practice and a good automatic camera M . L o c q u i n h a s e x t e n d e d t h i s t e c h n i q u e to
after a b l a n k test, o n e s o o n a c q u i r e s t h e k n a c k of o p t i c a l s e c t i o n s of v e r y s m a l l o b j e c t s , a n d to
s e l e c t i n g t h e r i g h t p a t t e r n of focal d i s t a n c e s . T h e e l e c t r o n m i c r o s c o p e s e c t i o n s . T h e c o n t o u r dif-
r e s u l t s o b t a i n e d a r e s u c h t h a t o n e m a y well f e r e n c e a d o p t e d is 1 to 2 , w h i c h m e a n s t h a t
68 Instruments and techniques

o n l y o n e of s e v e r a l s e c t i o n s is c h o s e n in t h e l a t t e r T i m e l a p s e p h o t o g r a p h y is of m o s t i n t e r e s t in
case. m i c r o s c o p y b e c a u s e g r o w t h in c u l t u r e m e d i a is
s l o w a n d it c a n b e a c c e l e r a t e d artificially, w h i c h
e n a b l e s o n e to d e t e c t p h e n o m e n a w h i c h t h e eye
Solid reconstructions
c a n n o t see. P i n o c y t o s i s h a s b e e n filmed b y M .
O n e can cut out the d r a w i n g s or photos as above Bessis in l e u c o c y t e s .
a n d stick t h e m to c a r d s ; t h e c a r d s h a v e b e e n c u t T o d e t e r m i n e t h e s p e e d a t w h i c h t h e film
t o t h e s a m e s h a p e s a n d a r e m a d e from m a t e r i a l should be m a d e , one measures the total d u r a t i o n
of t h e s a m e t h i c k n e s s a s t h e c o n t o u r i n t e r v a l of t h e m o v e m e n t s w h i c h a r e to b e filmed a n d
( d e p t h difference b e t w e e n s u c c e s s i v e s e c t i o n s , e s t i m a t e s t h e t i m e w h i c h t h e p r o j e c t e d film h a s
multiplied by the magnification used); one can t o r u n f r o m 20 s to 2 m i n . K n o w i n g t h a t t h e
s t a c k t h e s e c a r d s a n d r e c o n s t r u c t a m o d e l of t h e film is t o b e p r o j e c t e d a t a r a t e of 16 f r a m e s s" 1, if
original object three dimensionally. T h e s e o p - silent, o r a t 2 5 - 2 6 f r a m e s s" 1 if s o u n d o r T V
e r a t i o n s a r e facilitated if t w o o p p o s i t e c o l o u r e d film, o n e c a l c u l a t e s t h e n u m b e r of f r a m e s to b e
lines a r e i n c l u d e d w i t h i n t h e object, for e x a m p l e r e c o r d e d in this t i m e , a n d t h e n c a l c u l a t e s t h e
t w o f i l a m e n t s of g e l a t i n i m p r e g n a t e d w i t h I n d - s p e e d a t w h i c h to film.
i a n ink. T h e r e a r e n o w m a n y different t i m e l a p s e
d e v i c e s a v a i l a b l e . S o m e of t h e m p e r f o r m a c c o r d -
i n g to a p r e - s e t p r o g r a m m e : t h e y s w i t c h o n ,
Cine reconstructions
e x p o s e t h e film, a d v a n c e it, s w i t c h off t h e light,
W e h a v e seen h o w to r e c o n s t r u c t o b j e c t s from a n d r e p e a t t h i s cycle a t v a r i a b l e i n t e r v a l s . I f
r e a l s e c t i o n s o r from o p t i c a l s e c t i o n s p r o d u c e d e x p o s u r e s a r e close t o g e t h e r , t h e light is n o t
by drawings. O n e can semi-automate the proce-
d u r e by using two microscopes which are con-
nected by an image superimposition comparison
t u b e , b e h i n d w h i c h a film c a m e r a is p l a c e d . T h e
first s e c t i o n is p l a c e d o n m i c r o s c o p e A , p o s i -
tioned, held and photographed; without remov-
i n g t h e object, t h e s e c o n d p r e p a r a t i o n is p l a c e d
o n m i c r o s c o p e w h i c h is s u p e r i m p o s e d o n t h e
i m a g e of t h e first b y i l l u m i n a t i n g t h e t w o m i c r o -
s c o p e s ; m i c r o s c o p e A is t h e n s w i t c h e d off, a n d
t h e i m a g e is r e c o r d e d . T h e o p e r a t i o n is r e -
peated by placing a third preparation on micro-
s c o p e A a n d so o n . I t is p o s s i b l e to a n a l y s e , for
e x a m p l e , a v a s c u l a r s y s t e m a n d a l s o to p r o d u c e
t h r e e - d i m e n s i o n a l r e c o n s t r u c t i o n s after p r i n t i n g
o n p a p e r , w i t h o u t h a v i n g to d r a w t h e o b j e c t .
I n d e e d , o n e c a n s o m e t i m e s p r o d u c e a cine film if
o n e h a s e n o u g h s e c t i o n s ; this gives a d y n a m i c
i n s i g h t i n t o t h e w o r k i n g s of t h e object (Figure
1.68).

Cinemicrography
C i n e m i c r o g r a p h y h a s enjoyed r e v i v e d p o p u l a r -
ity w i t h t h e d e v e l o p m e n t of p h a s e c o n t r a s t
m i c r o s c o p y , w h i c h m a k e it p o s s i b l e to o b s e r v e
d e t a i l s w i t h i n t h e cell in vivo. J . D r a g e s c o , for
e x a m p l e , h a s e x p l o i t e d t h e s e possibilities in t h e
infusoria v e r y c o n v i n c i n g l y . A s in o r d i n a r y c i n e -
m a t o g r a p h y , o n e c a n film in r e a l t i m e a n d
project at the same speed, or one can use
F i g u r e 1.67. Cross-section through cinemicrographic
different s p e e d s a n d s p e e d u p o r slow d o w n t h e equipment with image projection accessory, and a general
m o v e m e n t s w h e n t h e film is p r o j e c t e d . view
Synthesis 69

Figure 1.68. T w o shots from a sequence, showing mitosis, and superimposition of a number on the
film. Top rightsuperimposed text on a film; lower rightsuperimposed clock. The instrument in
Figure 1.67 is employed for these effects

t u r n e d off b e t w e e n s u c c e s s i v e e x p o s u r e s (Figure Drawing at the m i c r o s c o p e


L68); 35 m m film, a s u s e d b y j . P a i n l e v e , y i e l d s
e x c e p t i o n a l l y g o o d r e s u l t s , b u t it is e x p e n s i v e ; T h e r e are three m e t h o d s used to d r a w at the
16 m m film is u s e d m u c h m o r e w i d e l y . T h e m i c r o s c o p e : (a) t h e g r a t i c u l e e y e p i e c e , (b) p r o -
G t t i n g e n I n s t i t u t e of C i n e m a t o g r a p h y ( G e r - j e c t i o n a n d (c) s u p e r i m p o s i t i o n of t w o i m a g e s
m a n y ) h a s a n e x h a u s t i v e c a t a l o g u e of s u c h films with the c a m e r a lucida or a d r a w i n g tube. T h e
a n d publishes a booklet entitled 'Research g r a t i c u l e e y e p i e c e m e t h o d is f a m i l i a r to all
Film'. artists w h o superimpose a graticule on a draw-
T h e c h o i c e of e m u l s i o n s u s e d in t h e p r o c e s s is i n g w h i c h t h e y w i s h to r e p r o d u c e . T h i s t e c h n i -
the s a m e as was discussed earlier u n d e r Photo- q u e is t e d i o u s a n d r e l a t i v e l y i n a c c u r a t e , b u t is
m i c r o g r a p h y , w i t h o n e difference; t h e e y e ' s t h e c h e a p e s t . P r o j e c t i o n o n to t h e w o r k t a b l e a t
c a p a c i t y to fuse i m a g e s , w h i c h a r e s e p a r a t e in t h e s i d e of t h e m i c r o s c o p e c a n b e u s e d , if t h e
t i m e , r e s u l t s in p r o j e c t e d p i c t u r e s w h i c h a r e r o o m is fairly d a r k ; t h e o u t l i n e s of t h e object a r e
a l w a y s s u p e r i o r to e n l a r g e d , fixed i m a g e s . p l o t t e d d i r e c t l y o n to t h e p a p e r ; it c a n a l s o b e
T h e p h o t o s e n s i t i v i t y of living o b j e c t s v a r i e s u s e d for d i r e c t r e c o r d i n g o n to p h o t o g r a p h i c
widely. O n e should always protect the speci- paper, producing a negative. Superimposing
m e n s from h e a t a n d u l t r a v i o l e t l i g h t as far a s t w o i m a g e s b y m e a n s of a n a p p r o p r i a t e o p t i c a l
p o s s i b l e , b y i n t e r p o s i n g effective u l t r a v i o l e t a n d d e v i c e , w h i c h w a s for a l o n g t i m e t h e c a m e r a
h e a t filters. lucida, has now become the drawing tube. This
70 Instruments and techniques

Figure 1.69. A mirror which has microlenses on its


surface rotates at high speed in the projected real image
plane to widen the exit pupil of the microscope and
cancel out the effects of internal defects in the eye
(Wild-Leitz, France, Rueil Malmaison)

F i g u r e 1.71. Drawing and projection mirror. This can only


be used in the dark, whereas the drawing tube in Figure
1.70 can be used in full daylight (Wild-Heerbrugg)

has m a d e work m u c h more comfortable, under


m o r e stable conditions. N e u t r a l grey filters
which are removable provide balanced adjust-
m e n t of i l l u m i n a t i o n of m i c r o s c o p e a n d p a p e r .
B i n o c u l a r o b s e r v a t i o n is p o s s i b l e w h e n t h e
d r a w i n g t u b e is u s e d ; t h i s c a n n o t b e d o n e w i t h
t h e c a m e r a l u c i d a (Figures 1.69-1.72).

Artefacts
A r t e f a c t s of m i c r o s c o p y a r e o b j e c t s artificially
c r e a t e d b y t h e o p e r a t o r d u r i n g t h e c o u r s e of
m a n i p u l a t i o n s w h e n p r e p a r i n g or examining
o b j e c t s . T h e r e a r e m a n y s o u r c e s of a r t e f a c t s ,
m a k i n g t h e i r d e t e c t i o n difficult. M u c h p r a c t i c e
is u s u a l l y r e q u i r e d to identify t h e m . A c c i d e n t a l
c o n t a m i n a t i o n is u s u a l l y t h e easiest to d e t e c t , as
it is n o t r e p r o d u c i b l e , w h e r e a s a r t e f a c t s d u e to
t h e t e c h n i q u e of p r e p a r a t i o n r e c u r u n t i l o n e
c h a n g e s t h e m e t h o d . T h i s is w h y a m i c r o s c o p i c
finding c a n n o t b e c o n s i d e r e d a s p r o v e n u n t i l it
Figure 1.70. Wild's microscope, fitted with a drawing tube h a s b e e n o b s e r v e d b y a t least t w o different
which projects the image of the paper surface on which the techniques. Certain constantly recurring arte-
drawing is to be made, inside the image tube. One can trace
facts a r e t a k e n to b e c h a r a c t e r i s t i c s of a s t r u c -
the outline of the object under observation
(Wild-Heerbrugg) ture.
Measurements 71

Figure 1.72. Comparison tubes (left) and demonstration tubes (right) (Leitz-Wetzlar)

1.6 Measurements MICROMETERS


O b j e c t m i c r o m e t e r s p r o v i d e a m e a n s of c a l i b r a t -
Measuring techniques
i n g m i c r o m e t e r e y e p i e c e s . T h e y a r e also u s e d to
For several centuries the microscope was an c a l i b r a t e all o t h e r s y s t e m s of l i n e a r m e a s u r e -
i n s t r u m e n t of q u a l i t a t i v e o b s e r v a t i o n . I t s n a t u - m e n t u s e d in t h e m i c r o s c o p e , b u t it is easiest to
r a l e v o l u t i o n w a s t h a t it w o u l d b e c o m e q u a n t i t a - u n d e r s t a n d h o w t h e y w o r k in c o n j u n c t i o n w i t h
tive in p a r a l l e l w i t h t h e sciences w h i c h u s e it. m i c r o m e t e r eyepieces. S o m e microscope m a n u -
S i n c e its d e v e l o p m e n t took p l a c e u n d e r t h e facturers have standardized their production
p r e s s u r e of n e e d s of t h e s e d i v e r s e s c i e n c e s , t h e r e a n d supply m i c r o m e t e r - m e a s u r i n g eyepieces,
w a s n o logical e v o l u t i o n of t h e m i c r o s c o p e a s a w i t h a f a c t o r w h i c h is r e l a t e d to t h e objectives
measuring instrument. Even nowadays, certain u s e d , t o r e a d d i m e n s i o n s d i r e c t l y in m i c r o -
p o t e n t i a l s r e m a i n r e s t r i c t e d b e c a u s e of t h e few m e t r e s . T h e s e m e a s u r e m e n t s should be check-
u s e r s in t h e n a r r o w fields for w h i c h t h e y w e r e e d a t l e a s t o n c e ; often, w h e n a d e s i g n is c h a n g e d ,
c r e a t e d . I n t h e c o n t e x t of g e n e r a l b i o l o g y t h i s t h e c a l i b r a t i o n is c h a n g e d w i t h o u t t h e m a n u f a c -
h a n d b o o k covers not only general m e a s u r i n g t u r e r b e i n g a w a r e of it, p a r t i c u l a r l y if t h e p a r t s
t e c h n i q u e s b u t also t h o s e s p e c i a l i z e d t e c h n i q u e s h a d b e e n p u r c h a s e d a t different t i m e s .
w h o s e u s e s a r e likely to e x p a n d in t h e f u t u r e . M i c r o m e t e r eyepieces or m e a s u r i n g eyepieces
s h o u l d b e c a l i b r a t e d n o t o n l y w i t h e a c h objec-
MEASURING LENGTHS tive, b u t also with a n d w i t h o u t each accessory,
C o n s i d e r a t i o n will n o t b e g i v e n h e r e to t h o s e w h o s e i n t e r p o s i t i o n c a n affect t h e l i g h t p a t h a n d
special microscopes which are designed as alter the overall magnification. T h e s e accessor-
m e a s u r i n g i n s t r u m e n t s , u s u a l l y in t h e m e t a l l u r - ies, s u c h a s b i n o c u l a r o r t r i n o c u l a r t u b e s , in-
gical o r i n d u s t r i a l s c i e n c e s . W e s h a l l d e a l h e r e termediate photographic tubes, extensible
o n l y w i t h t h o s e m e t h o d s w h i c h c a n b e a p p l i e d to t u b e s , d r a w i n g t u b e s , e t c . , all b e a r o n t h e i r
m e a s u r e l e n g t h s in a n y b i o l o g i c a l m i c r o s c o p e . housings the magnification which they produce.
I n s e l e c t i n g t h e m a g n i f i c a t i o n , o n e h a s to c o m - U s u a l l y t h i s is 1.25 o r 1.5, e x c e p t for z o o m
p r o m i s e b e t w e e n t w o conflicting c o n s i d e r a t i o n s : m a g n i f i c a t i o n s , w h i c h c a n r a n g e from 0.5 to 2.
if o n e selects a h i g h m a g n i f i c a t i o n , o n e loses O n e s h o u l d n o t u s e z o o m m a g n i f i e r s for m e a s u r -
definition a t t h e e d g e of s t r u c t u r e s b e c a u s e of i n g w i t h t h e m i c r o s c o p e , s i n c e o n e is n o t s u r e
diffraction fringes a n d t h e low c o n t r a s t of s o m e w h e r e o n e is o n t h e m a g n i f i c a t i o n r a n g e . O n e
o b j e c t s ; if o n e c h o o s e s l o w m a g n i f i c a t i o n , t h e c a n o n l y k n o w w h e r e o n e is b y o p e r a t i n g a t o n e
p i c t u r e lacks r e s o l u t i o n . I n p r a c t i c e , o n e a p p l i e s stop or the other, either m a x i m u m or m i n i m u m ,
t h e following s t a t i s t i c a l r u l e : m a g n i f i c a t i o n s b u t in e i t h e r c a s e o n e is u s u a l l y n o t w o r k i n g
g r e a t e r t h a n 1000 for all o b j e c t s s m a l l e r t h a n o p t i m a l l y for t h e o b j e c t i v e , p a r t i c u l a r l y if u s i n g
a b o u t , a b o u t 100 t i m e s for all o b j e c t s of a high p o w e r objective. O n e m a y m a k e inadver-
a b o u t 10 a n d , finally, m a g n i f i c a t i o n of less t e n t e r r o r s in m e a s u r e m e n t s u n l e s s o n e s t a n d a r -
t h a n 10 for o b j e c t s in t h e m i l l i m e t r e r a n g e .
dizes the operating conditions.
72 Instruments and techniques

C a l i b r a t i o n c o n s i s t s of c o m p a r i n g 1 o n t h e p a i r of m a r k s w h i c h c o i n c i d e s a s closely a s
o b j e c t scale w i t h 1 o n t h e m e a s u r i n g p o s s i b l e . If is t h e n u m b e r of g r a d a t i o n s of t h e
e y e p i e c e scale. T h e object m i c r o m e t e r , w h i c h is eyepiece micrometer which separates the two
u s u a l l y 1 m m d i v i d e d i n t o 100 d i v i s i o n s w i t h m a r k i n g s , a n d b t h e n u m b e r of g r a d a t i o n s o n
s m a l l e r s u b d i v i s i o n s a t o n e e n d , is p l a c e d o n t h e t h e o b j e c t m i c r o m e t e r scale, a n d i f / i s t h e v a l u e
s t a g e of t h e m i c r o s c o p e . T h e l i g h t i n g is a d j u s t e d in m i c r o m e t r e s of o n e d i v i s i o n o n this l a t t e r
to n o r m a l c o n d i t i o n s of o b s e r v a t i o n . Before fit- scale (usually s t a m p e d on the t u b e ) , the 'micro-
ting the m e a s u r i n g eyepiece into the microscope m e t r i c v a l u e ' m of t h e e y e p i e c e scale u s e d o n
t u b e , o n e s h o u l d e n s u r e t h a t t h e scale is well t h e s e e x a c t c o n d i t i o n s will b e
s u i t e d to t h e p a r t i c u l a r o b s e r v e r . If t h e eye h e
n o r m a l l y u s e s h a s a s t i g m a t i s m , his s p e c t a c l e s m = J
s h o u l d n o t b e r e m o v e d . I n all o t h e r c a s e s , t h e a
o b s e r v e r m a y t a k e off his glasses w i t h o u t affect- F o r e x a m p l e , if a g i v e n o p t i c a l c o m b i n a t i o n
i n g t h e a c c u r a c y of his r e a d i n g s (Figure 1.73). r e s u l t s in 20 d i v i s i o n s of 10 b e i n g e q u a l to 6 0
g r a d a t i o n s o n t h e e y e p i e c e scale, t h e n t h e
m i c r o m e t r i c v a l u e will b e 3.3 . T h e u s e r is
0 2 0 3 0 4 0 50> s t r o n g l y a d v i s e d to m a k e a t a b l e of t h e s e e q u i v a -
1 1
1 I I ' '
1 j
l e n c e s for all c o n d i t i o n s of o b s e r v a t i o n , m a r k b y
m a r k a n d for e a c h m a g n i f i c a t i o n , a n d p l a c e this
t a b l e in front of his m i c r o s c o p e . O b v i o u s l y ,
p a r a m e t e r s should not be changed between
c a l i b r a t i o n a n d m e a s u r i n g t h e l e n g t h of t h e
t u b e , t h e i n t e r m e d i a t e t u b e if fitted, t h e m a g n i -
fication a d j u s t m e n t , a n d of c o u r s e t h e o b j e c t i v e .
I f a n o b j e c t i v e is c h a n g e d s u b s e q u e n t l y , e v e n if
the n e w objective has the s a m e n o m i n a l m a g n i -
fication, the calibration m u s t be checked, a n d
c o r r e c t e d if n e c e s s a r y .
T h e s a m e m e t h o d of c a l i b r a t i o n c a n b e u s e d
w i t h a p r o j e c t i o n fitting o r a d r a w i n g e y e p i e c e
F i g u r e 1.73. Topeyepiece micrometer; b u t , a s in t h e c a s e of c a l i b r a t i n g a film, it is
belowcalibration of the eyepiece scale b e t t e r to d o w i t h o u t t h e e y e p i e c e scale a n d
with the object micrometer. At a always record on a separate small rule, on a
magnification of 500 in this example, 7 c o r n e r of t h e d r a w i n g o r a t t h e e n d of t h e film,
divisions of the eyepiece scale correspond
t w o s c a l e g r a d a t i o n s a t 10, 100 o r 1000 from
to two divisions ( = 20 ) on the object
micrometer; the value of one division on t h e object scale.
the eyepiece micrometer is thus 2.857 After t h e e y e p i e c e scale h a s b e e n c a l i b r a t e d ,
t h e size of t h e u n k n o w n object is m e a s u r e d b y
H a v i n g m a d e this a d j u s t m e n t , t h e m e a s u r i n g b r i n g i n g its e d g e t a n g e n t i a l to a l a r g e g r a d a t i o n
e y e p i e c e is i n t r o d u c e d a n d t h e o b j e c t m i c r o - o n t h e e y e p i e c e scale a n d c o u n t i n g t h e n u m b e r
m e t e r is b r o u g h t i n t o focus. T h i s is u s u a l l y d o n e of g r a d a t i o n s w h i c h s e p a r a t e it from t h e t a n g e n t
p h o t o g r a p h i c a l l y , a n d p r o d u c e s a c l o u d of silver g r a d a t i o n o r n e a r its o t h e r e n d ; o n e t h e n r e a d s it
grains, particularly at high magnification, with off from t h e t a b l e of e q u i v a l e n t s in m i c r o m e t r e s .
a d e n s i t y w h i c h d i m i n i s h e s t o w a r d s t h e e d g e of A l t e r n a t i v e l y , o n e c o u l d also p r o d u c e a scale
t h e g r a d u a t i o n m a r k , w h i c h limits t h e a c c u r a c y g r a d u a t e d in m i c r o m e t r e s for e a c h o p t i c a l c o m -
of c a l i b r a t i o n . By m o v i n g t h e e y e p i e c e , t h e t w o b i n a t i o n , a n d u s e t h i s for m e a s u r e m e n t s .
scales a r e m a d e p a r a l l e l a n d t h e n b y m o v i n g t h e T o improve measurements, some microscop-
s t a g e , t h e e n d m a r k of o n e of t h e scales is ists h a v e b e e n a d v o c a t i n g for a l o n g t i m e t h e u s e
b r o u g h t i n t o c o i n c i d e n c e w i t h t h e e n d scale of of s p e c i a l d r u m - t y p e m i c r o m e t e r e y e p i e c e s , in
t h e o t h e r . A t h i g h m a g n i f i c a t i o n , o n e of t h e t w o w h i c h a w i r e is d r i v e n b y a g r a d u a t e d k n u r l e d
will b e m u c h w i d e r t h a n t h e o t h e r a n d t h e m a r k screw, operated by the observer. T h e m o v e m e n t
o n t h e e y e p i e c e m i c r o m e t e r is a d j u s t e d to t h e of t h e w i r e c a n t h e n b e r e a d off, i n s t e a d of a
c e n t r e of t h e m a r k of t h e object m i c r o m e t e r . T h e s c a l e . E y e p i e c e s of t h i s t y p e a r e w i d e l y u s e d in
o b s e r v e r t h e n looks a t t h e scales to find t h e first industrial and metallurgical instruments, which
Measurements 73

a r e m u c h bigger a n d m o r e stable t h a n biological (2) T h e o b j e c t m a y b e d r a w n o r p h o t o g r a p h e d


microscopes. I n practice, unless one h a s a deli- at a s t a n d a r d magnification, then the out-
cate touch, the microscope tube undergoes elas- l i n e carefully c u t o u t a n d w e i g h e d o n a n
tic d e f o r m a t i o n w h e n t h e e y e p i e c e is h a n d l e d a c c u r a t e b a l a n c e ; a k n o w n a r e a of t h e s a m e
a n d a c c u r a c y suffers a s a r e s u l t . W i t h h i g h p a p e r in t h e s a m e h u m i d i t y is t h e n w e i g h e d ,
m a g n i f i c a t i o n s , t h i s loss offsets t h e g a i n m a d e after checking that the paper is
over a conventional biological microscope. Fit- h o m o g e n e o u s , b y t e s t - w e i g h i n g a n u m b e r of
ting an electronic sensor with separate reading r e c t a n g l e s of t h e s a m e a r e a . W h e n u s i n g t h e
on a milliammeter increases such a n error even p h o t o g r a p h i c m e t h o d , it is a d v i s a b l e to
m o r e . T h e r e f o r e , a c c e s s o r i e s of t h i s t y p e s h o u l d m a k e p o s i t i v e s o n reinforced s h r i n k - p r o o f
b e confined to v e r y rigid i n s t r u m e n t s , u s u a l l y of p a p e r , u s i n g t h e t e c h n i q u e s of p h o t o g r a m -
t h e r e v e r s e d i n d u s t r i a l t y p e o r to w o r k a t l o w metry and photographic map-making. Con-
magnifications. In the latter case the accuracy ventional papers have very variable shrink-
p o s s i b l e is l i m i t e d b y m a n y f a c t o r s . age coefficients and are not very
T o i m p r o v e t h e a c c u r a c y of r e a d i n g s , in s p i t e h o m o g e n e o u s in t h e d i r e c t i o n of t h e fibres
of lack of r i g i d i t y of t h e s t a n d , o r i n d e e d to a n d a t r i g h t a n g l e s to it.
m e a s u r e moving objects or those which h a v e (3) A n e y e p i e c e w i t h e n g r a v e d s q u a r e s is u s e d ,
complex outlines, two British companies have t h e n t h e n u m b e r of s q u a r e s c o v e r e d b y t h e
p r o d u c e d r e s o l v e d i m a g e s y s t e m s , in w h i c h t h e o b j e c t is c o u n t e d a n d t h e r e a d i n g c o n v e r t e d
t w o i m a g e s a r e in c o m p l e m e n t a r y c o l o u r s , into micrometres by applying the calibra-
u s u a l l y r e d a n d b l u e . T h e w h o l e field is r e s o l v e d t i o n factor. T h i s is t h e w o r s t m e t h o d b e -
so t h e o b s e r v e r o n l y h a s to b r i n g t w o different c a u s e , if t h e s q u a r e s a r e s m a l l , t h e y m a s k
coloured images into coincidence and then read t h e i m a g e a n d o n e m a k e s m i s t a k e s in c o u n t -
off t h e r e s o l u t i o n o n t h e k n o b of t h e d r i v e s c r e w ; i n g , w h e r e a s if t h e s q u a r e s a r e l a r g e , m a n y
t h e a n g l e of t h e s t a n d o r a n y m o v e m e n t of t h e s q u a r e s will o n l y b e p a r t i a l l y c o v e r e d b y t h e
o b j e c t h a s n o effect o n t h e r e a d i n g o r its a c c u r a - o b j e c t , t h u s c r e a t i n g a n o t h e r s o u r c e of e r r o r .
cy. (4) A r e a s c a n b e m e a s u r e d w i t h a n i n t e g r a t i n g -
T h e s e s y s t e m s a l s o p r o v i d e a m e t h o d of m a k - w h e e l p l a n i m e t e r ; t h e r e a r e t w o m e t h o d s of
i n g r a p i d c o u n t s in e a c h c a t e g o r y , o n e i t h e r side d o i n g t h i s , e i t h e r o n t h e p r o j e c t e d i m a g e (or
of p r e d e t e r m i n e d fixed v a l u e s b e c a u s e t a n g e n t indeed the drawing or photo) or on the
i m a g e s s t a n d o u t a n y w h e r e in t h e field. I t is a l s o i m a g e of t h e o b j e c t in t h e e y e p i e c e . T h e first
p o s s i b l e to m e a s u r e t h e e c c e n t r i c i t y of q u a s i - a l t e r n a t i v e is b y far t h e m o s t c o n v e n i e n t ,
circular objects by using a rotating stage or by accurate and cheap.
r o t a t i n g t h e e y e p i e c e . T h e s e a r e t h e o n l y t y p e s of
d e v i c e s u i t a b l e for v e r y s m a l l o b j e c t s , for d e t a i l s GRATICULES AND RETICULES
of o b j e c t s , o r t h o s e w i t h c o m p l i c a t e d o u t l i n e a n d G r a t i c u l e s a r e s y s t e m s of lines e n g r a v e d o n a
re-entrant angles. Even measurements on p l a n e g l a s s d i s c w h i c h is i n t e r p o s e d in t h e focal
p h o t o m i c r o g r a p h s are not as good, since the p l a n e of a n e y e p i e c e . T h e s y s t e m of lines m a y b e
e s t i m a t i o n of t h e p o s i t i o n of t h e e d g e s of a n of a n y d i m e n s i o n o r p a t t e r n . R e t i c u l e is t h e t e r m
o b j e c t d e p e n d s o n c o n t r a s t a n d t h e r e c i p r o c i t y of u s e d to d e s c r i b e t h e r e c t a n g u l a r g r i d s c o n s t i -
t h e e m u l s i o n in t h e d e v e l o p e r . t u t e d b y s u c h lines.
Figure 1.74 s h o w s s o m e t y p e s of g r a t i c u l e s a n d
M E A S U R E M E N T OF AREAS
reticules. T h e caption describes their applica-
T h e s e m e a s u r e m e n t s a r e c a r r i e d o u t b y elec- t i o n s . R e t i c u l e s c a n b e rigidly fitted, o r p l a c e d in
tronic means, using quantimeter microscopes a m o b i l e m o u n t i n g w h i c h is a d j u s t e d b y a
(see S e c t i o n 1.9, p . 9 0 ) . If t h e s e e x p e n s i v e i n s t r u - micrometer screw with a g r a d u a t e d barrel.
m e n t s a r e n o t a v a i l a b l e , t h e r e a r e four a l t e r n a - B a r r e l e y e p i e c e s c a n o n l y b e u s e d a t low to
tives: m e d i u m magnifications, or with instruments
(1) T h e o b j e c t m a y b e d r a w n a t a s t a n d a r d w h i c h h a v e v e r y s t a b l e s t a n d s . T h e y a r e excel-
m a g n i f i c a t i o n , t h e d r a w i n g c o p i e d o n to l e n t w h e n fitted to i n v e r t e d m i c r o s c o p e s w i t h
m i l l i m e t r e g r a p h p a p e r , t h e n u m b e r of massive stands. With simple direct microscopes,
squares within the outline counted, and the a m e r e t o u c h of t h e h a n d will r e s u l t in flexure of
reading converted into square micrometres t h e s t a n d , a m p l i f i e d b y t h e o p t i c a l lever a r m ,
after c a l i b r a t i o n . a n d this destroys the theoretical accuracy.
74 Instruments and techniques

Figure 1.74. Reticules and graticules. Cross-hairs for a polarization microscope. Double measuring scale.
Rectangular grid for counts

MEASURING VOLUMES AND WEIGHTS should be detected by carrying out successive


By superimposing drawings or p h o t o g r a p h s calibrations. For this reason, observers w h o
t a k e n a t p r e d e f i n e d , e q u a l i n t e r v a l s of d e p t h , o n m a k e f r e q u e n t m e a s u r e m e n t s of d e p t h prefer to
moving the micrometer screw appropriately, u s e s p e c i a l m i c r o s c o p e s fitted w i t h o n e , t w o o r
o n e c a n m a k e g r a p h i c r e c o n s t r u c t i o n s of t h e t h r e e v e r n i e r s w h i c h m e a s u r e t h e m o v e m e n t s of
s p e c i m e n o n a n e n l a r g e d scale. I t s w e i g h t c a n b e t h e s t a g e a n d h e n c e of t h e object d i r e c t l y . T h e
m e a s u r e d by weighing the p a p e r sections a n d
m u l t i p l y i n g t h e surface a r e a s b y a d e n s i t y factor,
e s t a b l i s h e d from t h e fresh o b j e c t o r from its d r y
weight. M . Locquin has extended these techni-
q u e s of g r a p h i c r e c o n s t r u c t i o n to m e t h o d s a s
s m a l l a s a few t e n s of m i c r o m e t r e s , b y d r a w i n g
optical r a t h e r t h a n microtome sections. Never-
t h e l e s s , t h i s m e t h o d is t e d i o u s a n d full of e r r o r s ,
if o n e w a n t s to m a k e a c c u r a t e d e t e r m i n a t i o n s of
m i n u t e objects.

M E A S U R E M E N T OF ANGLES
Angles can be measured either with a goniomet-
ric e y e p i e c e , o r b y u s i n g a r o t a t i n g s t a g e w h i c h
is g r a d u a t e d a n d a n e y e p i e c e w i t h c r o s s - h a i r s .
M e a s u r e m e n t s of this t y p e d o n o t p r e s e n t a n y
p a r t i c u l a r difficulties, u n l e s s a n a c c u r a c y g r e a -
t e r t h a n 1 is r e q u i r e d (Figure 1.75).

M E A S U R E M E N T OF D E P T H S
T h e d e p t h of a n o b j e c t d e n o t e s its t h i c k n e s s
a l o n g t h e o p t i c a l axis, i.e. p e r p e n d i c u l a r to t h e
p l a n e of o b s e r v a t i o n . T h e a c c u r a c y of t h e s e
m e a s u r e m e n t s is g o v e r n e d b y t h e u n c e r t a i n t y of F i g u r e 1.75. Goniometrie eyepiece, showing graticule.
t h e d e p t h of t h e reference p o i n t , since a t l e a s t The divided circle is fixed, while the cross-hairs can be
o n e face of t h e o b j e c t is b e i n g v i e w e d t h r o u g h its rotated. It is equally possible to measure angles with
the cross-hairs fixed and rotation of the stage of the
w h o l e t h i c k n e s s ; it is also i n f l u e n c e d b y t h e polarization microscope
refractive i n d e x of t h e object, t h e c a l i b r a t i o n of
t h e fine a d j u s t m e n t a n d t h e fine m o v e m e n t of operator measures positions a n d reads directly
t h e m i c r o s c o p e . T h e p r e c i s e d e t e r m i n a t i o n of in m i c r o m e t r e s after s u b t r a c t i n g t h e initial from
refractive i n d i c e s is d i s c u s s e d in d e t a i l o n p a g e t h e final n u m b e r .
9 2 . W h e n c a l i b r a t i n g t h e m i c r o m e t e r s c r e w , it F o r m e a s u r e m e n t s in d e p t h , t h e c o r r e c t i o n for
s h o u l d b e n o t e d t h a t this c o m p o n e n t is g e n e r a l l y t h e refractive i n d e x is as follows: w i t h a d r y
n o n - l i n e a r in o p e r a t i o n . Before t a k i n g a n y r e a d - objective, one multiplies the reading by the
i n g , t h e r e f o r e , a n y i r r e g u l a r i t i e s a l o n g its p a t h refractive i n d e x of t h e m o u n t i n g m e d i u m ; if
Special techniques 75

NlS

: Objective a n d t h e n u m b e r of p o l l e n s in t h e r e s i d u e is

NPv = NP

x /
1 / a n d t h e n u m b e r of p o l l e n s in 1 m l of initial
1 / s e d i m e n t is
E \ j / C
NPv
NP/m\ =
v. Cover-slip D'

D 1 DISSOCIATION
W h e n two s u p e r i m p o s e d objects c a n n o t be sepa-
rated by direct observation, one m u s t dissociate
Figure 1.76. The correction made necessary by
refraction when measuring the thickness of a t h e m , as one does with curves. I n order to
section using an air objective. The apparent p r e s e r v e o n e of t h e m , o n e s u b t r a c t s t h e o t h e r b y
distance, ED', is shorter than the real thickness, c o n t r o l l i n g its i n t e n s i t y r e l a t i v e to a s t a n d a r d .
ED F o r e x a m p l e , o n e c a n t a k e t w o p h o t o g r a p h s of
the s a m e section using the s a m e staining proce-
d u r e , before a n d after b l o c k i n g a p a r t i c u l a r
operating with i m m e r s i o n objective, one multi-
r e a c t i o n of i n t e r e s t . T w o serial s e c t i o n s c a n b e
plies b y t h e q u o t i e n t n/n', w h e r e is t h e refrac-
c u t , a n d t h e y a r e u s u a l l y sufficiently s i m i l a r to
tive i n d e x of t h e o b j e c t a n d ri t h e refractive
dissociate photographically by superimposing
i n d e x of t h e m o u n t i n g m e d i u m (Figure 1.76).
t h e n e g a t i v e of o n e c o n d i t i o n o n t h e p o s i t i v e of
t h e o t h e r , o n e of t h e m m a s k i n g t h e r e a c t i o n o n e
QUANTITATIVE COUNTING
w i s h e s to s u b t r a c t .
I n palynology one m u s t count the pollen types
q u a n t i t a t i v e l y . T h e n u m b e r of s p o r e s a n d p o l -
l e n s in a p a r t i c u l a r s a m p l e is fairly c o n s t a n t , b u t
v a r i e s m u c h from o n e s a m p l e to a n o t h e r . T h e s e 1.7 Special techniques
differences p e r m i t o n e to c h a r a c t e r i z e p h a s e s ,
e v e n if t h e r e is n o t m u c h difference in p e r c e n -
Locating objects
t a g e s of t y p e s . F r o m t h e b e g i n n i n g , o n e m u s t u s e
c o v e r - s l i p s of t h e s a m e t h i c k n e s s , to m a k e all t h e I t is e s s e n t i a l to b e a b l e to l o c a t e a n o b j e c t
o b s e r v a t i o n s a t t h e s a m e m a g n i f i c a t i o n , a n d to q u i c k l y in a p e r m a n e n t p r e p a r a t i o n . T h e p r i n -
s c a n t h e s a m e n u m b e r of lines in t h e s a m e w a y . c i p a l m e t h o d s in o r d e r of i n c r e a s i n g a c c u r a c y
O n e h a s to c a l i b r a t e t h e s y s t e m initially b y a r e a s follows: (a) If a single m i c r o s c o p e is b e i n g
w e i g h i n g a p r e c i s e v o l u m e of d i l u t e g l y c e r i n e u s e d , o r t w o m i c r o s c o p e s of t h e s a m e m o d e l , o n e
very accurately. T h e n one always uses a n iden- c a n l o c a t e t h e c o o r d i n a t e s b y r e f e r e n c e to t h e
tical s a m p l e of g l y c e r i n a t e d w a t e r i n t o w h i c h t h e v e r n i e r s of t h e s t a g e , b u t t h e s e c o o r d i n a t e s d o
spores or pollens are mixed. n o t a l w a y s h a v e t h e s a m e p o i n t of o r i g i n w i t h
I f F i s t h e i n i t i a l v o l u m e of t h e s a m p l e (in m l ) , different m o d e l s o r m a k e s , (b) O n e c a n d r a w a t
is t h e v o l u m e of t h e s p e c i m e n in g l y c e r i n e after l o w m a g n i f i c a t i o n o r p h o t o g r a p h t h e w h o l e field
t r e a t m e n t (in m l ) , S is t h e s u r f a c e a r e a of t h e o r o b j e c t a n d define t h e i n t e r e s t i n g d e t a i l s b y
2
c o v e r - s l i p (in c m ) , s is t h e s u r f a c e a r e a of t h e c o l o u r e d p o i n t s , (c) U s i n g a l o w p o w e r o b j e c -
2
l i n e a r r e g i o n e x p l o r e d (in c m ) , N[ is t h e n u m b e r t i v e , o n e c a n m a r k a c o l o u r e d s p o t w i t h a felt
of lines c o u n t e d , G is t h e v o l u m e of a d r o p (in p e n a t t h e s i d e of t h e o b j e c t . A l t e r n a t i v e l y , a
m l ) , Ng is t h e n u m b e r of d r o p s p l a c e d o n t h e finder can be inserted with n u m b e r e d boxes.
c o v e r - s l i p , is t h e v o l u m e of t h e d e p o s i t b e - T h i s d e v i c e e n a b l e s o n e to m a t c h t w o m i c r o -
t w e e n t h e c o v e r - s l i p a n d slide (in m l ) , a n d is photographs.
t h e t o t a l n u m b e r of s p o r e s a n d p o l l e n s c o u n t e d , N o n e of t h e m e t h o d s listed is a s g o o d a s a
t h e n t h e n u m b e r of p o l l e n s o n t h e c o v e r - s l i p is r e c e n t l y i n t r o d u c e d s y s t e m n o t y e t well k n o w n .
given by A fine p l a s t i c f i n d e r is a t t a c h e d to t h e b a c k of t h e
76 Instruments and techniques

slide w h i c h s u p p o r t s t h e object. T o define t h e


l o c a t i o n of a n object, a low p o w e r o b j e c t i v e is
m o u n t e d a n d t h e focus a d j u s t e d to t h e b a s e of
t h e slide. T h e c o o r d i n a t e s of t h e b o x e s c a n t h e n
b e r e a d off. T h e s e a r e fixed w i t h r e s p e c t to t h e
p r e p a r a t i o n , h e n c e t h e r e is n o d a n g e r of e r r o r ,
w h i l e t h e fact t h a t t h e g r i d is o u t s i d e t h e focal
p l a n e of t h e object m e a n s t h a t it d o e s n o t
interfere w i t h o b s e r v a t i o n of t h e object, as it
r e m a i n s c o m p l e t e l y invisible. H o w e v e r , e v e n
p e r m a n e n t o b j e c t s c a n m o v e u n d e r t h e influence
of g r a v i t y , especially if slides a r e s t a c k e d v e r t i -
cally, b e c a u s e t h e r e s i n s a r e n e v e r q u i t e solid,
a n d m a y r e m a i n v i s c o u s in t h e m i d d l e of t h e
field. F i g u r e 1.77. Perspective view of a Kfler heating and
cooling stage. A condenser is housed in the chamber. The
slide with the specimen is placed in the upper chamber,
Special stages and warmed or cooled stages closed by means of a plane cover-slip; a fluid of known
temperature indicated by a thermometer circulates in the
I n o r d e r to e x a m i n e a n object in its e n t i r e t y , o n e lower chamber
m u s t p r o v i d e m e a n s for s m o o t h a n d m e a s u r e d
m o t i o n in b o t h a x e s of t h e p l a n e of t h e s t a g e . T h e s e a c c e s s o r i e s a r e e x t r e m e l y useful for r e d u c -
W i t h m o v i n g m i c r o o r g a n i s m s , it is p r e f e r a b l e to i n g t h e t h i c k n e s s of a n object, e n h a n c i n g its
h a v e l u b r i c a t e d friction s t a g e s w h i c h c a n m o v e d e t a i l s , e x a m i n i n g its b e h a v i o u r u n d e r c r u s h i n g
in all d i r e c t i o n s , i n c l u d i n g in a c i r c u l a r m a n n e r forces, e t c . M o s t c o m p r e s s o r s a r e m e c h a n i c a l ,
or along the a n d y axes only. b u t w e prefer t h e Bessis a n d L o c q u i n d e s i g n
S y s t e m a t i c e x p l o r a t i o n , o r i g i n a l l y in o r d e r to w h i c h p r e s s e s t h e o b j e c t a g a i n s t t h e slide c o v e r
m a k e c o u n t s a n d t h e n for q u a n t i m e t r i c a n a l y s e s b y s w e l l i n g a t h i n film of a g a r a g a r o r g e l a t i n .
of i m a g e s , h a s p r o v i d e d a n i m p e t u s t o w a r d s t h e Slides a r e p r e p a r e d , i m p r e g n a t e d w i t h a 1 %
d e v e l o p m e n t of s t a g e s w i t h m o v e m e n t c o n t r o l - gelatin or agar agar solution a n d then dried.
led b y m i n i a t u r e m o t o r s , g i v i n g c o n t i n u o u s o r S p e c i m e n s a r e s u s p e n d e d in a v e r y s m a l l d r o p l e t
stepwise motion. Similarly, rotary stages, which of w a t e r a n d t h e n c o v e r e d w i t h a slide. T h e
a r e i n d i s p e n s a b l e for p o l a r i m e t r y , h a v e s o m e - w a t e r is a b s o r b e d a n d t h e s p e c i m e n s a r e c o m -
times been motorized. p r e s s e d b y t h e s w e l l i n g of t h e gel. O p e r a t i o n of
T h e s e s t a g e s all h a v e o n l y t w o o r t h r e e d e - conventional mechanical compressors can be
g r e e s of f r e e d o m . S o m e t i m e s in p o l a r i m e t r y o n e i m p r o v e d b y i n s e r t i n g a s m a l l p l a t e of glass
m u s t tilt t h e object w i t h r e s p e c t to t h e o p t i c a l u n d e r t h e slide, following t h e p r a c t i c e of Bessis
a x i s . T o a c h i e v e t h i s , t h e object is t r a p p e d a n d B r i c k a , so t h a t t h e s p e c i m e n s a r e c o m p r e s s -
b e t w e e n t w o glass h e m i s p h e r e s , t h r o u g h w h i c h e d in t h e c e n t r e of t h e field b y e l a s t i c d e f o r m a -
it is e x a m i n e d w i t h objectives w i t h l o n g focal tion.
l e n g t h s d e s i g n e d especially for this p u r p o s e .
T w o a d d i t i o n a l d e g r e e s of f r e e d o m a r e i m p a r t e d VERNIERS
in a d d i t i o n to m o v e m e n t s a r o u n d t h e a n d y T h e s e d e v i c e s a r e s m a l l r u l e d scales, c a l i b r a t e d
a x e s . S t a g e s of this d e s i g n a r e called t h e o d o l i t e o v e r a l e n g t h of 9 m m . T h e 9 m m a r e d i v i d e d
stages. i n t o 10 e q u a l s p a c e s , e a c h o n e 0.9 m m in l e n g t h .
Stages with heating or cooling accessories are T h e r e is t h u s a difference of 0.1 m m b e t w e e n
u s e d in m i c r o c h e m i s t r y for m e a s u r e m e n t s of t h e s e scale d i v i s i o n s a n d a w h o l e m i l l i m e t r e
d e p r e s s i o n of m e l t i n g p o i n t s o r s u b l i m a t i o n d i v i s i o n . O n e c a n t h u s m e a s u r e t e n t h s of a
t e m p e r a t u r e s (Figure 1.77). m i l l i m e t r e b y c o m p a r i n g t h e v e r n i e r scale
S o m e s t a g e s h a v e p r o v i s i o n for p r o d u c i n g a a g a i n s t a n o r d i n a r y m i l l i m e t r e scale. T h e n u m -
v a c u u m s u r r o u n d i n g t h e object in o r d e r to b e r of t h e m i l l i m e t r e scale w h i c h c o m e s closest
b o m b a r d it w i t h p a r t i c l e s , for s t r i p p i n g off t h e t o t h e z e r o p o i n t o n t h e v e r n i e r scale is first r e a d
s u r f a c e , o r for p r o d u c i n g f l u o r e s c e n c e . T h e s e a r e off. T h e n u m b e r of g r a d a t i o n s o n t h e v e r n i e r
n o t w i d e l y u s e d in biology. s c a l e a r e t h e n c o u n t e d u n t i l a m a r k is f o u n d
M i c r o c o m p r e s s o r s c a n b e fitted to o v e r s t a g e s . which coincides with a m a r k on the millimetre
Special techniques 77

s c a l e , for e x a m p l e , t h e f o u r t h m a r k . S i n c e t h e r e aberration, the two images being synthe-


is a difference b e t w e e n o n e d i v i s i o n o n t h e s i z e d t h r o u g h a n a n a m o r p h o s i s g r i d ( 1969).
m i l l i m e t r e scale a n d o n e d i v i s i o n o n t h e v e r n i e r (5) C o n t r o l of i m a g e a n a l y s i s b y s c a n n i n g t h e
s c a l e , t h e first four m a r k s , 0, 1, 2 a n d 3 a r e 0.4, image, either detecting discontinuities or
0 . 3 , 0.2 a n d 0.1 m m , r e s p e c t i v e l y , from a m a r k h o m o g e n e i t i e s in t h e i m a g e , u s i n g q u a n -
o n the millimetre scale. O n e can thus m e a s u r e timetric microscopes.
t h e p o s i t i o n of a n o b j e c t to w i t h i n 0.1 m m o n t h e (6) C o n t r o l of t h e i m a g e from its d e r i v a t i v e s o r
two axes. i n t e g r a l s , to p r o d u c e e l e c t r o n i c i m a g e m o d i -
fiers of t h e p h a s e c o n t r a s t o r i n t e r f e r e n c e
t y p e s , p r o v i d i n g a g r e a t e r r a n g e of possibili-
Microscopic observations u n d e r increased
ties ( 1 9 7 0 ) .
pressure
(7) C o n t r o l l i n g i l l u m i n a t i o n a t a c o n s t a n t level
G a v a u d a n et al. s h o w e d t h a t it is p o s s i b l e to b y m e a n s of p h o t o s e n s i t i v e filters.
operate a microscope and examine specimens (8) C o n s t a n t t e m p e r a t u r e c o n t r o l of t h e object.
u n d e r h i g h p r e s s u r e s . T h e front lenses of h i g h (9) C o n t r o l l i n g a flashlight to give c o n s t a n t
p o w e r objectives h a v e very small surface areas, levels o n t h e film.
hence they can withstand considerable pres-
2
s u r e s , s u c h a s 100 k g . c m " if t h e y a r e well se- S o m e of t h e s e d e v i c e s h a v e n o t y e t r e a c h e d t h e
cured. O n l y the objective forms a n integral p a r t c o m m e r c i a l s t a g e , b u t will c e r t a i n l y d o so w h e n
of t h e c h a m b e r u n d e r p r e s s u r e . I t is p o s s i b l e to t h e p r o d u c t i o n cost falls c o m p a r e d to t h e cost of
e x a m i n e living o b j e c t s u n d e r h i g h p r e s s u r e s . c o n v e n t i o n a l m e a n s . I t s h o u l d b e n o t e d t h a t all
t h e s e a p p l i c a t i o n s of a u t o m a t i o n a r e m u c h
e a s i e r w i t h p l a n e m e t a l l i c surfaces t h a n w i t h
Automation in m i c r o s c o p y b i o l o g i c a l o b j e c t s , w h i c h a r e less r e g u l a r in
shape and have m u c h weaker contrast.
P r o g r e s s t o w a r d s a u t o m a t i o n is a c o m m o n fea-
t u r e in all fields. I t w a s first a p p l i e d to a c c e s s o r - L i n e a r , s t e p w i s e o r s i n u s o i d a l s c a n n i n g of
ies before b e i n g u s e d in t h e m i c r o s c o p e itself, a s i m a g e s b y m e c h a n i c a l m o v e m e n t of t h e s t a g e
it is n a t u r a l l y e a s i e r to a u t o m a t e e l e c t r o m e c h a n - u n d e r t h e c o n t r o l of s e r v o m o t o r s is a p u r e l y
ical s y s t e m s t h a n o p t i c a l o n e s . T h u s , t h e m i c r o - mechanical operation which has been auto-
tome, dehydrating systems, embedding, stain- m a t e d for a l o n g t i m e .
ing systems a n d differentiation have been wholly S o m e a u t o m a t e d o p e r a t i o n s of a c c e s s o r y
c r p a r t l y a u t o m a t e d , w i t h o u t t o o m u c h difficul- e q u i p m e n t of t h e m i c r o s c o p e s h o u l d also b e
ty. T h e s e d e v i c e s a r e d e s c r i b e d in t h e a p p r o p r i - m e n t i o n e d , s u c h a s a u t o - c o n t r o l of t h e v i d i c o n
a t e s e c t i o n s of this b o o k . C e r t a i n o t h e r o p e r a - o r o r t h o c o n i m a g e s of television c a m e r a s , e.g.
t i o n s in t h e m i c r o s c o p e , h o w e v e r , h a v e o n l y auto-adjustment by varying the optical density
b e e n a u t o m a t e d fairly r e c e n t l y a n d t h i s h a s of s e c t o r s of t h e i l l u m i n a t i o n of t h e sensitive
s o m e t i m e s b e e n v e r y e x p e n s i v e in c a p i t a l . T h e s u r f a c e s of c i n e c a m e r a s .
following a r e e x a m i n e d in g r e a t e r d e t a i l in t h e O v e r a l l , w e still a r e far from t h e m i c r o s c o p e
a p p r o p r i a t e sections. w h i c h a u t o m a t i c a l l y e x a m i n e s in s e q u e n c e all
t h e o b j e c t s p r e s e n t e d to it, selects p r e v i o u s l y
(1) A u t o m a t i o n of p h o t o m i c r o g r a p h i c c a m e r a s , d e f i n e d o b j e c t s for c o u n t i n g o r p h o t o g r a p h y , o r
with automatic exposure time and advance calls t h e o b s e r v e r to e x a m i n e t h e m . C o m b i n a -
of t h e film a t t h e e n d of e x p o s u r e . t i o n of k n o w n m e t h o d s , e l e c t r o n i c , e l e c t r o -
(2) A u t o m a t i c c o n t r o l of c o n s t a n t level of illu- optical or photometric, with existing knowledge
m i n a t i o n , i n c l u d i n g c o r r e c t i o n for v a r i a - in p h o t o n m i c r o s c o p y , s h o u l d e v e n t u a l l y l e a d to
t i o n s in t h e o p a c i t y of a n o b j e c t , as d e - t h i s . T h e o v e r a l l m e t h o d s a p p l i e d b y L o c q u i n in
v e l o p e d b y L o c q u i n in 1959. t h e ' E c c e n t r o n ' a r e e v i d e n c e of t h e c e r t a i n t y of
(3) M e c h a n i c a l a d j u s t m e n t of focusing of t h e such development.
o b j e c t i v e to t h e a v e r a g e focal p l a n e of t h e
object (1968).
Microsurgery
(4) O p t i c a l c o n t r o l to t h e a v e r a g e p l a n e of t h e
i m a g e , i n d e p e n d e n t l y of m o v e m e n t s of t h e MICROPROBES
o b j e c t , o r c h a n g e s in its s h a p e ; t h i s is d o n e M i c r o p r o b e is a g e n e r a l t e r m u s e d to c o v e r all
by a compromise between a sharp image d e v i c e s w h i c h a c t o n a h i g h l y localized r e g i o n of
a n d a n i m a g e w h i c h is b l u r r e d b y s p h e r i c a l t h e o b j e c t in o r d e r to i n t e r v e n e m i c r o s u r g i c a l l y .
78 Instruments and techniques

T h e first t y p e of m i c r o p r o b e is t h e u l t r a f i n e Microprobes to d e s t r o y intracellular


m e c h a n i c a l tool w h i c h is u s e d m a n u a l l y , s o m e - o r g a n e l l e s w i t h a r u b y l a s e r w e r e first u s e d b y
t i m e s fitted w i t h a d e l a y , w i t h a m i c r o d i s s e c t o r , Bessis in 1 9 6 1 ; t h e b e a m e n t e r e d t h e e y e p i e c e of
o r w i t h a m i c r o m a n i p u l a t o r . T h e m a i n tools a r e t h e m i c r o s c o p e to b e focused b y t h e o p t i c a l t r a i n
d e s i g n e d to p i n , p i e r c e , a p p l y m e c h a n i c a l o r o n to a m i n u t e p o i n t o n t h e object. A n a u x i l i a r y
e l e c t r i c a l s t i m u l i , c a u t e r i z e , inject, h o l d , o r li- aiming device indicated the exact point. T h i s
g a t e cells o r p a r t s of cells. T h e s e c o n d t y p e uses m i c r o p r o b e w a s l a t e r u s e d , after i n c r e a s i n g its
e l e c t r i c c u r r e n t to c a u t e r i z e , c u t , c o n d u c t c u r - p o w e r , to m a c h i n e m e t a l s o r e x a m i n e t h e m
rent a n d perform micro-electrophoresis or s p e c t r o g r a p h i c a l l y . T h e m o s t c o m m o n solid-
rheophoresis, among other operations. s t a t e l a s e r s n o w in u s e a r e t h e r u b y - n e o d y m i u m
T h e t h i r d t y p e m a k e s p o i n t lesions b y a c t i n i c d o p e d g l a s s , w h i c h e m p l o y l i q u i d d y e s for t h e
light. C h a k h o t i n w a s t h e first to i n t r o d u c e u l t r a - trigger switch).
violet m i c r o p u n c t u r e . T h e a d v e n t of l a s e r s a n d Microtorches are based on the same principle
t h e i r a p p l i c a t i o n b y Bessis to d e s t r o y i n t r a c e l l u - b u t u s e a c o n t i n u o u s l a s e r , e i t h e r of h e l i u m -
lar organelles constituted a great advance. R u b y neon, argon or yttrium-aluminium-garnet
a n d n e o d y m i u m l a s e r s w e r e followed b y c o n - m i x t u r e ; t h e y c a n b e in t h e o r g a n i c o r l i q u i d
t i n u o u s e m i s s i o n l a s e r s , u s i n g visible o r i n f r a r e d p h a s e . L i q u i d p h a s e lasers are the only kind
l i g h t . T h e o l d e r t y p e s a r e p r e f e r a b l e in c y t o l o g y , w h i c h a l l o w t h e w a v e l e n g t h to b e v a r i e d c o n -
t h e l a t t e r in h i s t o l o g y , a s c o n t i n u o u s e m i s s i o n t i n u o u s l y . I n biology this is u s e d t o m a k e a s o r t
lasers are the only ones which can cut u p a n of m i c r o p u n c t u r e , s i m i l a r to t h a t w h i c h C h a k h o -
o b j e c t of c o m p l e x s h a p e efficiently. F u r t h e r - tin developed with the ultraviolet b e a m . H o w -
m o r e , t h e i n t e n s i t y of t h e r a d i a t i o n e m i t t e d e v e r , t h e l a s e r is m u c h m o r e flexible a n d offers
can be readily controlled a n d exposure times g r e a t e r possibilities. A m o n g the applications
c a n b e v a r i e d . T h e r e s t r i c t e d c h o i c e of a v a i l a b l e listed a b o v e , o n e m e r i t s s p e c i a l m e n t i o n t h e
w a v e l e n g t h s w a s a n o b s t a c l e to t h e u s e of l a s e r s h o l o g r a p h i n v e n t e d b y D . G a b o r in 1948; it
in b i o l o g y for a l o n g t i m e ; l o n g w a v e l e n g t h s w a s u s e d in m i c r o s c o p y before l a s e r s h a d b e e n
red a n d infraredwere usually used. T h e ready discovered. It h a s not yet been applied on a
a v a i l a b i l i t y of m o l e c u l a r l a s e r s w i t h d y e s h a s l a r g e scale to t h e m i c r o s c o p i c i m a g e , b u t h a s a
r e m o v e d t h i s o b s t a c l e . S o o n it will b e p o s s i b l e to very promising future.
u s e all w a v e l e n g t h s in t h e visible s p e c t r u m .
All t h e s e t y p e s of p r o b e h a v e b e e n p r o d u c e d MICROMANIPULATION AND MICRODISSECTION
with the special accessories with which they Micromanipulators are instruments which can
form a n assembly, such as m i c r o m a n i p u l a t o r s , operate at m e d i u m a n d high magnifications
laser microperforators, inverted microscopes, w i t h a single o b j e c t i v e . M i c r o d i s s e c t o r s w o r k a t
a n d ultraviolet microscopes. l o w m a g n i f i c a t i o n s in s t e r e o s c o p i c m i c r o s c o p e s
or binocular magnifying glasses.
M I C R O P R O B E S A N D LASER T O R C H E S
W h e n choosing a micromanipulator, one has
T h e h i g h d e g r e e of c o h e r e n c e , l o w d i s p e r s i o n
t o c o n s i d e r t h e t y p e of o p e r a t i o n . I t is e a s i e r to
a n d h i g h e n e r g y c o n t e n t of a l a s e r b e a m m a k e
s e c u r e a n o b j e c t in p o s i t i o n if t h e i n s t r u m e n t h a s
t h i s n e w s o u r c e a n e w tool w h e n c o m b i n e d w i t h
m e c h a n i c a l l y i n d e p e n d e n t m o v e m e n t s in t h e
t h e m i c r o s c o p e in a d d i t i o n to its u s e in o b s e r v a -
t h r e e c o o r d i n a t e s , w h e r e a s for m i c r o s u r g i c a l o p -
tion:
e r a t i o n s , it is m u c h b e t t e r to h a v e a single
(1) A p u l s e d l a s e r c a n b e u s e d to m a k e a m o v e m e n t d r i v e of t h e s c a n n i n g - s w e e p t y p e , a s
micropuncture, produce microcoagulation, fitted to t h e d e F o n b r u n e o r M o n c h a b l o n i n s t r u -
microdestruction, micro-evaporation and m e n t s . A c h a n g e of r e d u c t i o n r a t i o is n e c e s s a r y
microperforation, etc. w h e n t h e m a g n i f i c a t i o n is c h a n g e d . T h e d i r e c -
(2) A c o n t i n u o u s l a s e r c a n b e u s e d for m i c r o - t i o n of m o v e m e n t h a s to b e r e v e r s e d in c o n v e r t -
cutting, microsurgery, microseparation, i n g from a n e r e c t s t e r e o s c o p i c i m a g e m i c r o s c o p e
m i c r o - R a m a n s p e c t r o s c o p y , a s t u d y of t h e t o t h e i n v e r t i n g single o b j e c t i v e t y p e .
T y n d a l l effect a t t h e m i c r o s c o p i c level, I t t a k e s p r a c t i c e a n d m u c h p a t i e n c e to l e a r n
microcoagulation, microtracing, microholo- micromanipulation. O n the other hand, prfab-
graphy, photochemical activation on the r i c a t i o n of m i c r o - i n s t r u m e n t s l e n d s itself to s t a n -
m i c r o s c a l e , e x a m i n i n g t h e p h o t o t r o p i s m of dardization, or indeed to automation. T h e r e are
unicellular organisms or phototropism with- j i g s for a u t o m a t i c d r a w i n g of m i c r o p i p e t t e s ,
in a cell, e t c . microneedles a n d microsyringes. T h e ideal a n d
Use of monochromatic light 79

F i g u r e 1.78. A glass sleeve with an


inner surface coated with sterilized
glycerol for fine aseptic procedures.
The whole assembly is sterilized en bloc
before being used warm. This
assembly allows the use of a
stereoscopic microscope (de Fonbrune)

Figure 1.79. Microsphere and microhemisphere at the tip of a tube. Rightthe heating filament as
seen in the eyepiece of the de Fonbrune microforge; the same views are featured in all the following
figures up to Figure 1.91, and all operations are shown as visualized in the eyepiece of the de Fonbrune
microforge

c o m p r e h e n s i v e i n s t r u m e n t is t h e d e F o n b r u n e l i g h t , in t h e visible u l t r a v i o l e t a n d i n f r a r e d
m i c r o f o r g e . All t h e c o n t r o l s n e c e s s a r y for h e a t - r e g i o n s of t h e s p e c t r u m . M o n o c h r o m a t o r s s u i t -
ing a n d cooling are provided. T h e de F o n b r u n e a b l e for m i c r o s c o p y p r o v i d e a b e a m w h i c h is
m e t h o d s of f o r m i n g e s s e n t i a l tools a r e s h o w n in g e o m e t r i c a l l y fixed in s p a c e , b u t w h o s e
Figures 1.79-1.91. T h e following is a list of t h i s w a v e l e n g t h c a n b e v a r i e d . F o u r t y p e s of e q u i p -
s o r t of o p e r a t i o n : m a k i n g a c a l i b r a t e d m i c r o - m e n t can be used:
s p h e r e , d r a w i n g m i c r o n e e d l e s , b e n d i n g b y dif-
(1) D i s p e r s i o n filters ( C h r i s t i a n s e n ) , w h o s e
ferential h e a t i n g , d r a w i n g micropipettes, form-
w a v e l e n g t h is v a r i e d b y v a r y i n g t h e t e m p e r -
i n g a n orifice, c u p p i n g g l a s s e s , m a k i n g m i c r o -
ature.
s c a l p e l s , m a k i n g slides w i t h c u r v e d e d g e s . A
(2) L y o t p o l a r i z i n g filters, w h i c h a r e excellent,
c o l l e c t i o n of s u c h tools is e s s e n t i a l .
but expensive.
(3) C a t a d i o p t r i c m o n o c h r o m a t o r s (Figure 1.92).
(4) I n t e r f e r e n c e m o n o c h r o m a t o r s w i t h a F a b r y -
1.8 Use of monochromatic Perrot wedge standard.

light T h e l a t t e r t w o a r e t h e o n l y p r a c t i c a l o n e s for u s e
in microscopy. C a t a d i o p t r i c m o n o c h r o m a t o r s
a r e u s e d w h e n a c c u r a t e definition of t h e
Monochromators
w a v e l e n g t h is r e q u i r e d ; i n t e r f e r e n c e m o n o c h r o -
M o n o c h r o m a t o r s p r o v i d e a m e t h o d of c o n - mators are used when equipment must be com-
t i n u o u s l y v a r y i n g t h e w a v e l e n g t h s of a b e a m of p a c t ; in t h i s c a s e , t h e n a r r o w n e s s of t h e p a s s
80

Figure 1.80. Microneedles. Topdrawing; belowthree different forms

F i g u r e 1.81. Method of bending a glass rod by unequal


heating

F i g u r e 1.82. Bending glass


tubes, thick (upper) and
thin (lower)
81

F i g u r e 1.83. Making a bend in


order to draw a micropipette

F i g u r e 1.84. Breaking a glass thread


under remote heating. Leftwithout
cooling; rightair cooling

F i g u r e 1.85. Diameters of micropipettes drawn at the


same temperature, plotted as a function of the applied
1 2 3 4 5 6 7 8 9 11 12 1314 is 17./<m weight used for drawing (de Fonbrune)
82

F i g u r e 1.86. Drawing ultrafine micropipettes


smaller than 1 tip diameter

F i g u r e 1.87. Making calibrated


micropipettes, with straight and bent ends

F i g u r e 1.88. Making a side tube in a glass wall


83

F i g u r e 1.89. Making a cupping glass from blown


glass

Figure 1.90. Schouten's method of making a glass microscalpel

Figure 1.91. Making a glass slide to prevent crushing of microorganisms


84 Instruments and techniques

is e m i t t e d is t h e n p a s s e d t h r o u g h t h e m i c r o s c o p e
a n d e n t e r s a s p e c t r o g r a p h for a n a l y s i s .
T h e spark can be generated by an electric
c u r r e n t o r b y a p u l s e d laser. T h i s l a t t e r m e t h o d
is m u c h b e t t e r b e c a u s e as it is m u c h m o r e
l o c a l i z e d it r e q u i r e s less m a t e r i a l a n d t i m e . T h e
l a s e r is g e n e r a l l y a solid b a r , w i t h a p a s s i v e
t r i g g e r i n g d e v i c e (Q s w i t c h ) w h i c h amplifies t h e
p e a k p o w e r . T h e e n e r g y of t h e l a s e r is u s e d
almost completely to vaporize the small a m o u n t
of s u b s t a n c e a n a l y s e d ; h e n c e it is n e c e s s a r y to
h a v e a n a u x i l i a r y d e t o n a t o r w h i c h raises t h e
t e m p e r a t u r e of t h e g a s p r o d u c e d to ionize it to
generate the characteristic emissions with
a d e q u a t e intensity.
T h e microscope should have mirror or quartz
o p t i c s to m a k e it s u i t a b l e for u l t r a v i o l e t light.
T h i s microspectrograph can be used to analyse
Figure 1.92. Direct beam monochromator (Leitz-Wetzlar)
in cells a n d tissues t h e following e l e m e n t s o t h e r
t h a n c a r b o n a n d v e r y light e l e m e n t s w h i c h a r e
m o s t r e a d i l y d e t e c t e d : A g , A u , Al, A s , , B a ,
b a n d c a n b e i n c r e a s e d , w h i l e fixing it in s p a c e C a , C d , C o , Cr, Cs, C u , Fe, H g , , Li, M g , ,
b y h e a d - t o - t a i l m o u n t i n g of t w o i d e n t i c a l w e d g e N a , , P b , P t , S b , Si, S n , Sr, T i , T l , Z n . T h e
filters. a d v e n t of t h e s c a n n i n g e l e c t r o n m i c r o s c o p e w i t h
A m o n o c h r o m a t i c i n t e r f e r e n c e filter is in itself i n t e g r a l a n a l y s e r s h a s r e d u c e d t h e a p p e a l of
a s o r t of m o n o c h r o m a t o r , since it a l l o w s a s m a l l m i c r o s p e c t r o g r a p h s to a g r e a t e x t e n t .
v a r i a t i o n of t h e w a v e l e n g t h of t h e p a s s b a n d b y T h e r e a d e r is r e f e r r e d to S e c t i o n 1.11 for m o r e
c h a n g i n g t h e a n g l e of i n c i d e n c e of t h e b e a m . I t d e t a i l s o n t h e a d v a n t a g e s of t h e s e m e t h o d s . T h e
should be m o u n t e d on a calibrated rotary head o n e d r a w b a c k of e l e c t r o n m i c r o s c o p e s is t h a t t h e
to a c h i e v e this effect. s p e c i m e n s h a v e to b e d e h y d r a t e d a n d e x a m i n e d
u n d e r high v a c u u m .

Spectrographs
F o r a long time, the preferred m e t h o d has been
Microphotometers and
t o a n a l y s e t h e light after it h a s t r a v e r s e d t h e
microspectrophotometers
o b j e c t , r a t h e r t h a n i l l u m i n a t e t h e object w i t h
v a r i a b l e m o n o c h r o m a t i c light. M o n o c h r o m a t o r s T h e s e devices are microscopes which have been
w e r e p l a c e d after t h e m i c r o s c o p e a n d t h e i m a g e fitted w i t h v e r y s e n s i t i v e a n d a c c u r a t e p h o t o -
of t h e o b j e c t w a s p r o j e c t e d o n to t h e slit of t h e e l e c t r i c d e t e c t o r s w h i c h m e a s u r e t h e light in-
spectrograph. M i n i a t u r e eyepiece spectro- t e n s i t y , p o i n t b y p o i n t , o v e r t h e object. W h e n
g r a p h s , w h i c h w e r e fitted i n s t e a d of a n o r m a l t h e a p p a r a t u s is c o n n e c t e d to a n i n f o r m a t i o n
eyepiece, were marketed. T h e s e devices have p r o c e s s i n g u n i t , it b e c o m e s a q u a n t i m e t r i c
now practically disappeared, because the struc- m i c r o s c o p e (see S e c t i o n 1.9, p . 9 0 ) .
t u r e of t h e i m a g e b l u r s t h e p r o j e c t i o n of t h e M i c r o p h o t o m e t r i c r e a d i n g s p r o v i d e t h e fol-
s p e c t r u m . Nevertheless, these eyepiece spectro- lowing information a b o u t a n object: optical den-
s c o p e s a r e i n v a l u a b l e for s e l e c t i n g c o l o u r filters, sity to t r a n s m i t t e d l i g h t , e x t i n c t i o n of p o l a r i z a -
d e t e c t i n g t h e lines in a s p e c t r u m a n d c o n d u c t i n g t i o n , d e g r e e of c o n t r a s t in i n t e r f e r e n c e c o n t r a s t ,
c o a r s e a n a l y s e s of fluorescence light. reflective p o w e r , i n t e n s i t y of diffusion, r e -
e m i s s i o n (diffuse reflection), f l u o r e s c e n c e , T y n -
d a l l effect, e t c .
Spark microspectroscopes and e m i s s i o n
W h e n t h e i n s t r u m e n t is a t t a c h e d to a
spectrography
m o n o c h r o m a t o r with a continuously adjustable
I n p r i n c i p l e , a s p a r k h e a t s a p o i n t in t h e o b j e c t w a v e l e n g t h , it is c o n v e r t e d i n t o a m i c r o s p e c -
o b s e r v e d to a h i g h t e m p e r a t u r e ; t h e light w h i c h trophotometer.
Use of monochromatic light

MEASURING OPTICAL DENSITY


I n all c a s e s , o p t i c a l d e n s i t y is t h e p h o t o m e t r i c
v a r i a b l e m e a s u r e d , i r r e s p e c t i v e of w h e t h e r t h e
s p e c i m e n h a s its o w n i n t r i n s i c selective a b s o r p -
t i o n b a n d o r is s t r a i n e d specifically, o r w h e t h e r
t h e p r o d u c t i o n of t h e i m a g e is p h o t o m e t r i c a l l y
b o u n d u p w i t h t h e c h a r a c t e r i s t i c s of t h e o b j e c t .
T o obtain such measurements the microscope
is fitted w i t h a p h o t o m u l t i p l i e r , in series w i t h a
selective d i a p h r a g m r e s t r i c t i n g t h e a r e a to b e
measured. A n auxiliary illumination device dis-
p l a y s this d i a p h r a g m , s u p e r i m p o s e d o n t h e i m -
a g e of t h e o b j e c t , in o r d e r to select t h e p o i n t a t
w h i c h t h e r e a d i n g is to b e m a d e , a n d d e t e r m i n e
its d i a m e t e r (Figure 1.93).. G e n e r a l l y s p e a k i n g ,
a n i n t e r f e r e n c e filter o r o t h e r t y p e c a n b e i n -
s e r t e d after t h e d i a p h r a g m , in o r d e r to l i m i t t h e
s p e c t r a l p a s s b a n d . W h e n t h e i n s t r u m e n t is
e q u i p p e d to m a k e u l t r a v i o l e t r e a d i n g s , t h e
photomultiplier has a quartz window.
T h e p h o t o m u l t i p l i e r is a p h o t o e l e c t r i c cell
w h i c h c o n t a i n s a series of e l e c t r o n m u l t i p l y i n g
e l e c t r o d e s , to m u l t i p l y t h e e l e c t r o n s o r i g i n a l l y
d e t a c h e d b y t h e i m p a c t of p h o t o n s o n t h e p h o t o -
c a t h o d e . T h e s u p p l y v o l t a g e h a s to b e s t a b i l i z e d ,
b o t h a t t h e level of t h e s o u r c e of i l l u m i n a t i o n of
t h e m i c r o s c o p e a n d of t h e p h o t o m u l t i p l i e r . All
photomultipliers h a v e b a c k g r o u n d noise or d a r k
current, a n d they should not be o p e r a t e d at low
levels of i l l u m i n a t i o n w i t h o u t a l l o w i n g for it. I n
v i e w of t h e v e r y h i g h sensitivity of t h e s e i n s t r u -
ments, they should be used with d i m room
l i g h t i n g to a v o i d s t r a y light.
T h e a c c u r a c y of r e a d i n g s c a n b e e n h a n c e d b y
u s i n g a n e l e c t r o - o p t i c a l m o d u l a t o r s u c h as t h e
J e n a Velomet. T h e m o d u l a t o r contains a special
p e n u m b r a l l a m i n a which oscillates. T h e s e
o s c i l l a t i o n s modify t h e i n t e r f e r e n c e s t a t e a t t h e
specific p o i n t to b e e x a m i n e d . C o n v e r s i o n i n t o
a n electrical s i g n a l is effected b y a p h o t o m u l t i -
plier. After a p p r o p r i a t e p r o c e s s i n g in a n elec-
t r o n i c u n i t , t h e s i g n a l is d i s p l a y e d o n a m e t e r
with a centrally placed needle. F i g u r e 1.93. Leitz microphotometer. Path of
photons: 1, source; 2, collector; 3, field stop;
M E A S U R E M E N T S OF W A V E L E N G T H 4, aperture stop; 5, condenser; 6, specimen holder
W e h a v e s h o w n h o w t h e a d d i t i o n of a and specimen; 7, objective; 8, rear focal plane;
9, eyepiece diaphragm; 10, eyepiece; 11, beam
monochromator can convert a microphotometer
splitter; 12, trihedral mirror; 13, focal plane of
into a microspectrophotometer. If the finder eyepiece; 14, entry pupil of eye, and eye;
m o n o c h r o m a t o r is a n i n t e r f e r e n c e w e d g e - t y p e 15, lens; 16, measuring diaphragm;
filter, it is u s u a l l y m o u n t e d after t h e d i a p h r a g m 17, retractable mirror; 18, pilot lamp; 19, diffuser;
w h i c h selects t h e field to b e m e a s u r e d . If a p r i s m 20, photomultiplier. This novel system of
viewfinding enables the operator to project on to
o r r u l e d t y p e is u s e d , it is u s u a l l y p l a c e d b e - the object the diaphragm which defines the
tween the source and the specimen. In the latter measuring field, before making the measurement
c a s e , critical l i g h t i n g is u s e d ; t h e i m a g e of t h e (Leitz-Wetzlar)
86 Instruments and techniques

e m e r g e n c e slit of t h e m o n o c h r o m a t o r is p r o - t h e r e m a y b e insufficient cells to c o u n t . G o o d


j e c t e d o n to t h e s p e c i m e n b y a c o n d e n s e r . c o u n t i n g s h o u l d yield v a l u e s w h i c h a r e r e p r o -
S o m e i n s t r u m e n t s h a v e v a r y i n g d e g r e e s of d u c i b l e a n d a c c u r a t e to w i t h i n 3 % .
a u t o m a t i o n such as spectral absorption curve
p l o t t i n g w i t h c o n t i n u o u s s c a n n i n g of t h e s p e c - BLOODCOUNTS
t r u m , or p h o t o m e t r i c section t h r o u g h the object T h i s a p p l i c a t i o n is t h e m o s t c o m m o n . A p i p e t t e
a t a fixed w a v e l e n g t h . T h i s l a t t e r is k n o w n a s a w i t h a s m a l l b u l b w i t h m a r k s a t 1, 2 a n d 100, is
t o m o p h o t o m e t e r ( L o c q u i n ) . I n all cases it is u s e d for t h i s p u r p o s e . W h e n b l o o d is s a m p l e d ,
a d v i s a b l e to p h o t o g r a p h t h e i m a g e of t h e o b j e c t u p to t h e ' d i v i s i o n , t h e final d i l u t i o n is 1 % ;
w i t h t h e selection d i a p h r a g m in p o s i t i o n . w h e n u p to t h e ' 2 ' d i v i s i o n , t h e d i l u t i o n will b e
I n fluorescence m i c r o s c o p y , if t h e i n s t r u m e n t 1/200. T h e d i l u e n t is M a r c a n o ' s fluid: 5 g of
h a s g o o d filters, g l o b a l m e a s u r e m e n t of t h e light s o d i u m s u l p h a t e + 1 m l of formol 4- 0.01 g of
e m i t t e d is u s u a l l y a d e q u a t e . W h e r e s e v e r a l c o m - m e t h y l e n e b l u e a n d 100 m l w a t e r . C o u n t i n g is
p o n e n t s a r e p r e s e n t in t h e s a m e s t r u c t u r e , d o n e a t a m a g n i f i c a t i o n of X 200 to X 4 0 0 . W h i t e
h o w e v e r , it m a y b e useful to m a k e f u r t h e r blood corpuscles are rfringent a n d stain blue;
s e l e c t i o n of w a v e l e n g t h s b y u s i n g m o n o c h r o m a - e r y t h r o c y t e s a r e g r e y i s h (Figures 1.94 a n d 1.95).
tic i n t e r f e r e n c e filters. W h e n t h e s e i n s t r u m e n t s

m
a r e c o n n e c t e d to i n f o r m a t i o n p r o c e s s i n g u n i t s ,
they b e c o m e q u a n t i m e t r i c microscopes or
microquantimeters.

msmmmmm
1.9 Stereological
microanalysis
Counting
mm.
F i g u r e 1.94. Appearance of a
O n e h a s to d i l u t e a s p e c i m e n to p r o d u c e suffi-
rectangle in a Malassez cell, with
c i e n t s e p a r a t i o n , in o r d e r to d e t e r m i n e t h e e x a c t normal blood diluted 1/400
n u m b e r of o b j e c t s in s u s p e n s i o n in a b i o l o g i c a l
o r o t h e r fluid. T h e d i l u e n t m a y also h a v e a Before i n t r o d u c i n g t h e d i l u e n t i n t o t h e c o u n t -
n u m b e r of o t h e r f u n c t i o n s , s u c h as p r e s e r v a t i o n , i n g cell, t h e m i x t u r e s h o u l d b e h o m o g e n i z e d b y
s t a i n i n g a n d selective lysis of s o m e e l e m e n t s . r o t a t i n g t h e p i p e t t e v e r t i c a l l y for 2 m i n . A s m a l l
T h e s u s p e n s i o n is d i l u t e d in s p e c i a l c a l i b r a t e d g l a s s b e a d i n s i d e it stirs t h e c o n t e n t s . R e t i c u l o -
pipettes a n d transferred into a counting c h a m - c y t e s a r e c o u n t e d after h a e m o l y s i s w i t h a n e q u a l
b e r . T h i s c h a m b e r is a c a l i b r a t e d cell o n t h e v o l u m e of t h e following a g e n t : 1 g of cresyl
b a s e of w h i c h t h e r e is a g r a t i c u l e . T h e s e cells a r e b r i l l i a n t b l u e X 0.8 g of s o d i u m c h l o r i d e a n d
k n o w n b y v a r i o u s n a m e s , from t h e s y s t e m of 100 m l of w a t e r , filtered a t t h e t i m e of u s e . A t
calibration: Malassez, Thoma, Brker, least 2 0 0 - 3 0 0 corpuscles should always be
3
N e u b a u e r , e t c . ; t h e y give s i m i l a r r e s u l t s . C a r e c o u n t e d a n d t h e c o u n t referred to 1 m m by
s h o u l d a l w a y s b e t a k e n to e n s u r e t h a t t h e s p e c i a l m u l t i p l y i n g t h e figure b y 1 0 0 0 0 , in t h e c a s e of a
slide c o v e r w h i c h closes t h e c o u n t i n g c h a m b e r is 1 % d i l u t i o n (Figure 1.96).
p r e s s e d well h o m e o n its s u p p o r t , o r t h e r e will N o d i s t i n c t i o n s h o u l d b e m a d e in a c o u n t i n g
be large errors. cell b e t w e e n t h e v a r i o u s t y p e s of f o r m e d ele-
E r r o r s c a n also b e d u e to p o o r s t a n d a r d i z a - m e n t s , other t h a n erythrocytes a n d total white
t i o n of d i l u t i o n s o r of t h e d e p t h of t h e cells; cells. Before m a k i n g a t o t a l w h i t e cell c o u n t , t h e
c o v e r - s l i p s m a y n o t b e perfectly h o r i z o n t a l , t h e p e r c e n t a g e s of t h e v a r i o u s t y p e s a r e d e t e r m i n e d
assembly m a y have been poorly washed, sam- f r o m a s t a i n e d s m e a r (Figure 1.97). E o s i n o p h i l s ,
p l i n g of t h e s p e c i m e n m a y h a v e b e e n defective, h o w e v e r , c a n b e d e t e c t e d in t h e c o u n t i n g cell if
partial evaporation m a y have occurred, the they are stained bright red with Randolph's
s p e c i m e n m a y lack h o m o g e n e i t y , t h e o b s e r v e r s o l u t i o n . ( T h i s c o n s i s t s of e q u a l p a r t s , m i x e d a t
m a y b e t i r e d , t h e r e m a y b e u n c e r t a i n t i e s in t h e t h e t i m e of u s e , of t w o s o l u t i o n s : A , 0 . 1 % of
p o s i t i o n o r i d e n t i f i c a t i o n of c e r t a i n f e a t u r e s , o r m e t h y l e n e b l u e in 5 0 m l of p r o p y l e n e glycol a n d
Stereological microanalysis 87

-3mm

1 mmJ N e u b a u e r grid Trk grid


Thoma grid

-3mm-

1
2mm
-__4 0.05
70.20
^0.05

L_.T0.25

I
I
Brker grid
Malassez grid

F i g u r e 1.95. Main types of graticules for counting chambers

IE 0
olol
F i g u r e 1.96. Path followed when
counting. Erythrocytes which lie on F i g u r e 1.97. A differential leucocyte
the first of the triple ruled lines are count is obtained by counting 100
only counted on sides A and leucocytes on each side of a smear

50 m l of w a t e r ; B , 0 . 1 % of p h l o x i n e in 50 m l (a) fit t h e r u b b e r to t h e t i p a n d b l o w o u t t h e
p r o p y l e n e glycol a n d 50 m l of w a t e r . r e s t of t h e d i l u t e d b l o o d ;
Platelet counts are obtained u n d e r phase con- (b) d r a w u p the acetic acid;
t r a s t , after h a e m o l y s i s of t h e e r y t h r o c y t e s in t h e (c) w a s h w i t h distilled w a t e r s e v e r a l t i m e s ;
following s o l u t i o n : 3 g of c o c a i n e h y d r o c h l o r i d e (d) w a s h t w o o r t h r e e t i m e s w i t h a b s o l u t e e t h a -
a n d 0.2 g s o d i u m c h l o r i d e in 100 m l of w a t e r . nol;
CLEANING OF PIPETTES (e) w a s h w i t h e t h e r t w o o r t h r e e t i m e s , a n d let
P i p e t t e s a r e c l e a n if t h e glass b e a d d o e s n o t stick it flow o u t w i t h o u t b l o w i n g ;
t o t h e w a l l . I m m e d i a t e l y after c o u n t i n g , t h e r e - (f) l e a v e to d r y o r insufflate h o t a i r w i t h a
fore, it is e s s e n t i a l to: double bulb.
88 Instruments and techniques

I f a series of c o u n t s is b e i n g m a d e , it is
a d e q u a t e to w a s h t h e p i p e t t e w i t h distilled w a t e r
two or three times, a n d then with the diluent
t w i c e . O n e s h o u l d n e v e r let b l o o d d r y i n s i d e t h e
p i p e t t e ; if it h a p p e n s , t h e c a p i l l a r y s h o u l d b e
c l e a n e d w i t h h o r s e h a i r o r a little glacial a c e t i c
acid d r a w n u p a n d then w a s h e d as described
a b o v e , after d i s s o l v i n g t h e clot; b e t t e r still, o n e
c a n d i s s o l v e a clot b y d i g e s t i o n a t 37 C w i t h a
s o l u t i o n of 0.1 g of p e p s i n in 100 m l of 1 %
hydrochloric acid. T h e g r a d u a t e d pipettes are
w a s h e d w i t h distilled w a t e r a n d t h e n w i t h e t h a -
nol.

Morphometry and stereology


I n view of t h e b i o l o g i c a l v a r i a t i o n s of a t y p i c a l
b i o l o g i c a l object, it h a s l o n g b e e n a c k n o w l e d g e d
t h a t s t a t i s t i c a l m e t h o d s a r e n e c e s s a r y to assess
v a l i d i t y . H o w , t h e n , c a n o n e c o n v e r t r e a d i n g s of
F i g u r e 1.98. In a preparation which is viewed in plan, the
lengths into areas or volumes a n d with h o w probability of a segment of a straight line intersecting an
m u c h a c c u r a c y ? S u c h a r e t h e p r o b l e m s of m o r - object is proportional to the ratio of the diameter ofthat
p h o m e t r y o r m e a s u r e m e n t s of forms a n d of its object to the diameter of the field in a perpendicular
m a i n a s p e c t , s t e r e o l o g y , or t h e s t u d y of direction (Wild-Heerbrugg)
volumes.
L e t us a s s u m e t h a t a m i c r o s c o p i c object c o n -
t a i n s w i t h i n its v o l u m e g r a n u l e s of u n e q u a l size,
A a n d B . W h e n o n e c u t s a s e c t i o n of its v o l u m e
w i t h a m i c r o t o m e , t h e surface of t h e s e c t i o n
m u s t first of all b e of a d e q u a t e d i m e n s i o n s to
c o n t a i n e n o u g h r e p r e s e n t a t i v e e l e m e n t s of e a c h
c o n s t i t u e n t if s u b s e q u e n t s t a t i s t i c a l a n a l y s i s is to
h a v e a n y significance. W h e r e o p e r a t i n g c o n d i -
t i o n s a l l o w , r e a d i n g s m a d e in a n u m b e r of
s u c c e s s i v e s e c t i o n s a r e a d d e d u p to satisfy t h i s
condition.
E x p e r i m e n t a l analysis has yielded two princi-
ples of p r o p o r t i o n a l i t y :

(1) I n a t h i n s e c t i o n of a d e q u a t e surface a r e a ,
t h e a r e a s of s e c t i o n s of c o n s t i t u e n t s A a n d
will b e p r o p o r t i o n a l to t h e v o l u m e s o c c u -
pied by these constituents within the total
volume.
(2) I n a series of s e v e r a l t h i n s e c t i o n s of t h e Figure 1.99. Stereological analysis: a volume F is analysed
into planes which contain objects A in a matrix
same volume, the same applies. (Wild-Heerbrugg)
I t c a n b e d i r e c t l y inferred t h a t if a s e c t i o n , o r
series of s e c t i o n s , is of sufficient a r e a , t h e d e t e r - m e t h o d is b a s e d o n r e c u r r e n c e a n a l y s i s : if it is
m i n a t i o n of v o l u m e s is r e d u c e d to t h e d e t e r - p o s s i b l e to d e d u c e a v o l u m e from a n a r e a , it
m i n a t i o n of r e l a t i v e a r e a s . T h e e x a c t m e a s u r e - s h o u l d b e p o s s i b l e also to d e d u c e t h e a r e a b y
m e n t of a r e a s c a n b e d o n e b y t h e m e t h o d s l i n e a r o r p o i n t a n a l y s i s (Figures 1.98 a n d 1.99).
g i v e n in S e c t i o n 1.6, p . 7 3 . H o w e v e r , all of t h e s e L e t u s c o n s i d e r a section in a s p e c i m e n w i t h
m e t h o d s a r e t e d i o u s . T h i s is w h y a s i m p l e r a n d the two components A and occupying equal
more practical method has been sought. This p o r t i o n s of t h e a r e a of t h e field o b s e r v e d . A p o i n t
Stereological microanalysis 89

t a k e n a t r a n d o m in this field will h a v e e q u a l o c c u p i e d b y a n o b j e c t of u n i q u e n a t u r e , b u t


p r o b a b i l i t i e s of falling o n A o r o n B . T h i s d i s t r i b u t e d r a n d o m l y in s h a p e , a v e r a g e v o l u m e
p r o b a b i l i t y is t h u s 5 0 % for e a c h p o r t i o n . S i m p l e o r d i l u t i o n . C o u n t i n g of e r y t h r o c y t e s is a s i m p l e
r e a s o n i n g will d e m o n s t r a t e t h a t if t h e r a t i o s a n d p a r t i c u l a r c a s e of t h i s .
b e t w e e n A a n d d o n o t e x c e e d 1/2, a n d a r e W h e n o n e c u t s s e r i a l s e c t i o n s of a solid o b j e c t
g i v e n a v a l u e or y, t h e p r o b a b i l i t y of a g i v e n in e a c h s e c t i o n , p a r t i c l e s will b e p r e s e n t in
p o i n t b e i n g in A o r in will b e in t h e p r o p o r t i o n v a r y i n g s h a p e s a n d d i m e n s i o n s , s i n c e t h e sec-
orjv, o r w i t h t h e o t h e r , of its r e c i p r o c a l . T h i s t i o n w o u l d t r a v e r s e t h e l a r g e r d i a m e t e r of t h e
r e a s o n i n g c a n b e r e a d i l y e x t e n d e d to m o r e t h a n constituent object, only as dictated by statistics.
t w o c o m p o n e n t s o r to m o r e t h a n o n e single I n a section, the probability that an exploratory
p o i n t , if t h e s e p o i n t s a r e sufficiently d i s t a n t . s t r a i g h t line w o u l d i n t e r s e c t w i t h s u c h a n object
A p r i n c i p l e of m e a s u r e m e n t t h u s e m e r g e s , is p r o p o r t i o n a l to t h e d i a m e t e r of t h e o b j e c t a n d
w h i c h c a n b e f o r m u l a t e d a s follows. T h e d i s - t o t h e d i a m e t e r of t h e s e c t i o n in a d i r e c t i o n
t r i b u t i o n of t h e c o m p o n e n t s of a t h i n s e c t i o n of p e r p e n d i c u l a r to t h e s w e e p . I f t h e s w e e p lines
a n object in t h e m i c r o s c o p e in c a t e g o r i e s w h i c h are multiplied, one also multiplies the probabil-
a r e r e p r e s e n t a t i v e of t h e i r r e s p e c t i v e v o l u m e s , ity of i n t e r s e c t i o n . I t h a s b e e n d e m o n s t r a t e d
r e q u i r e s o n e o n l y to define, o n a p r e - e s t a b l i s h e d t h a t , w i t h t h e e x c e p t i o n of s p h e r i c a l b o d i e s , t h e
g r i d of e q u i d i s t a n t p o i n t s , t h e n u m b e r of p o i n t s r e l a t i o n b e t w e e n t h e c o u n t e d n u m b e r of in-
l o c a t e d o n e a c h c o m p o n e n t . T h i s c o u n t is p r o - t e r s e c t i o n s a n d t h e a r e a o c c u p i e d b y t h e object
p o r t i o n a l to t h e a r e a s a n d h e n c e to t h e r e l a t i v e o n t h e s e s e c t i o n s d e p e n d s o n t h e f o r m of its
v o l u m e s of t h e c o m p o n e n t s . m e a n v o l u m e a n d t h e o r i e n t a t i o n of t h e object.
I n p r a c t i c e , t h e o b s e r v i n g t u b e of a p o i n t R e t i c u l e s of s i n u s o i d a l s h a p e s h a v e b e e n d e -
r e t i c u l e e y e p i e c e u s u a l l y h a s b e t w e e n 25 a n d 42 s i g n e d to e l i m i n a t e t h e o r i e n t a t i o n effect, e.g. t h e
points. T h e accuracy can be improved by re- g r a t i c u l e . T h e i n f l u e n c e of t h e s h a p e of t h e
p e a t i n g t h e c o u n t in s e v e r a l fields in t h e s a m e p a r t i c l e o n t h e m e a s u r e m e n t s h a s b e e n ex-
s e c t i o n o r in s e v e r a l fields in s e v e r a l s u c c e s s i v e a m i n e d , e i t h e r b y c a l c u l a t i o n w h e r e its f o r m is
s e c t i o n s . I n t h i s c a s e t h e t o t a l n u m b e r of p o i n t s g e o m e t r i c a l l y s i m p l e o r b y e x p e r i m e n t if its form
is e q u a l to t h e n u m b e r of p o i n t s p e r field, is c o m p l e x , b u t s u c h a n e x p e r i m e n t is often
m u l t i p l i e d b y t h e n u m b e r of fields c o u n t e d . A difficult t o c a r r y o u t .
s i m p l e r u l e of p r o p o r t i o n a l i t y defines t h e r a t i o s . I n all m e a s u r e m e n t s of t h i s t y p e , a s in all
A c a l c u l a t i o n of e r r o r s is a d v i s a b l e to e n s u r e c o u n t i n g , o n e h a s to a l l o w for t a n g e n t i a l c o n -
t h a t a d e q u a t e n u m b e r s of r e p r e s e n t a t i v e p o i n t s t a c t s , b u t o n l y in t w o d i r e c t i o n s of t h e four
h a v e b e e n c o u n t e d for c o m p o n e n t s p r e s e n t in p o s s i b l e d i r e c t i o n s , p a r a l l e l to t h e e d g e s of t h e
low p e r c e n t a g e . F o r f u r t h e r d e t a i l s t h e r e a d e r is s q u a r e e x p l o r e d . T h e r e a d e r is r e f e r r e d to m o r e
referred to t h e i n s t r u c t i o n s for t h e u s e of i n t e g r a - s p e c i a l i z e d p a p e r s for f u r t h e r s t u d y . A n excel-
t o r s , o r to s p e c i a l i s t l i t e r a t u r e o n s t a t i s t i c s . l e n t g e n e r a l p r e s e n t a t i o n of t h e q u e s t i o n w a s
T a b l e s give t h e r e s u l t s of c a l c u l a t i o n of v a r i a n c e , m a d e b y A . S c h a e f e r in ' M i c r o s c o p i o n ' ( M a r c h
in p e r c e n t a g e , a s f u n c t i o n s of t h e n u m b e r of 1 9 7 0 ) , w i t h a r e f e r e n c e list of t h e e s s e n t i a l
p o i n t s m e a s u r e d , a n d t h e p r o p o r t i o n a l i t y of t h e l i t e r a t u r e u p to t h a t d a t e .
test p o i n t s o n t h e c o m p o n e n t s a n a l y s e d . W e Microstereology c a n be c o n d u c t e d purely by
s h a l l n o t e briefly t h a t 8 0 r e a d i n g s a r e r e q u i r e d m a n u a l m e a n s . T h e o p e r a t o r selects a field a n d
t o o b t a i n a s c a t t e r of less t h a n 5 % , if t h e c o u n t s by categories, type or density, the objects
p r o p o r t i o n of a c o m p o n e n t is 0.9, 150 if it is 0.6 w h i c h are located u n d e r the points, or which are
a n d 3 0 0 0 r e a d i n g s if it is 0 . 1 . A p r a c t i c a l r u l e i n t e r s e c t e d b y s e g m e n t s of t h e r e t i c u l e . H o w e v -
c a n b e d e r i v e d from t h i s . O n e h u n d r e d r e a d i n g s er, h e m a y u s e a p p a r a t u s of v a r y i n g d e g r e e s of
in a c o m p o n e n t a r e g e n e r a l l y a d e q u a t e if a n a u t o m a t i o n . T h e first s t e p t o w a r d s a u t o m a t i o n
a c c u r a c y of t h e o r d e r of 5 % is a c c e p t a b l e , in a is a u t o m a t i c s t a g e a d v a n c e . T w o m i c r o m o t o r s
c o m p o n e n t w h i c h is p r e s e n t a t 1 % . T h i s m e a n s , with reduction gearing drive the and y move-
of c o u r s e , 5 % of 1 % . m e n t s of t h e s t a g e . A s p e c i a l s w i t c h q u a n t i f i e s
the m o v e m e n t s by m o v i n g the p r e p a r a t i o n by
PARTICLES O F I D E N T I C A L T Y P E B U T O F V A R I E D t h e d i a m e t e r of a field e a c h t i m e t h e o p e r a t o r
FORM r e q u i r e s it. A s e c o n d s t a g e in a u t o m a t i o n is to
T h i s c o m m o n c a s e l e a d s d i r e c t l y to a u t o m a t i c r e p l a c e t h e e y e b y a p h o t o c e l l for c o u n t i n g . T h e
c o u n t i n g o r to a n a l y s i s of a r e a s o r of v o l u m e s microscope then becomes a microquantimeter.
90 Instruments and techniques

A n e x t r e m e l y c o m p r e h e n s i v e a n d refined i n s t r u - (xvii) a n a l y s i s of t h e c o m p l e m e n t of i n c o m p l e t e
m e n t of t h i s t y p e h a s b e e n d e v e l o p e d p a r t i c u l a r - p o r t i o n s of i m a g e s , o r ' c l o s i n g ' ;
ly for m e t a l l u r g i c a l a p p l i c a t i o n , b u t t h e r e a r e (xviii) Golay's transformation;
also m a n y biological uses at present, a n d (xix) i n p u t i n t o m e m o r y for s u b s e q u e n t c o m -
p e r h a p s t h e r e will b e m o r e in t h e f u t u r e . parison;
(xx) iteration on grey values;
(xxi) s c o r p o i d a n a l y s i s of c o n t o u r s b y a r e c -
Microquantimeters tangular or hexagonal matrix;
(xxii) defining positional anisotropics;
Q u a n t i m e t e r microscopes, or microquanti-
(xxiii) classification by nucleocytoplasmic
meters, carry out automated morphometric and
ratios;
stereological operations. I n principle, they con-
(xxiv) a u t o m a t i c t r a c i n g of e q u i d e n s i t o m e t r i c
sist of m i c r o p h o t o m e t e r s o r m i c r o s p e c t r o p h o t o -
contours;
m e t e r s in c o m b i n a t i o n w i t h a d a t a p r o c e s s i n g
(xxv) contrast reversal;
u n i t w h i c h c a n c o n d u c t t h e following o p e r a -
(xxvi) p l o t t i n g a n d t r a n s f e r of c o o r d i n a t e s ;
t i o n s , d e p e n d i n g o n its d e g r e e of c o m p l e x i t y a n d
(xxvii) d i g i t a l i z a t i o n of a n i m a g e in u p to 6 4
programme:
levels of g r e y .
() t e l e t y p e p r i n t - o u t of t h e m a t r i x r e p r e -
senting optical densities, with zig-zag ULTRASONIC MICROSCOPE
s c a n n i n g of t h e o b j e c t s ; R e c e n t l y it h a s b e c o m e p o s s i b l e to c o n s t r u c t
(ii) t h r e e - d i m e n s i o n a l r e p r e s e n t a t i o n of t h e l e n s e s w h i c h refract u l t r a s o u n d , f o r m i n g i m a g e s
o p t i c a l d e n s i t i e s r e a d o u t in s u c h a s c a n - w i t h a m a g n i f i c a t i o n of XlOO. T h e i n t e r e s t of
ning operation; t h i s n e w m i c r o s c o p e is t h a t it c a n form i m a g e s of
(Hi) s t a t i s t i c a l a n a l y s i s a n d classification of ' t h i c k ' s e c t i o n s of m a t e r i a l s , like m e t a l s a n d
l e n g t h s , s u r f a c e a r e a s a n d v o l u m e s , after n u m e r a l s , w h i c h a r e t r a n s p a r e n t to u l t r a s o u n d .
s e l e c t i o n from l i n e a r d i m e n s i o n s , o p t i c a l T h i s i n s t r u m e n t h a s a p p l i c a t i o n s in m i c r o -
density, particle s h a p e , etc.; palaeontology.
(iv) s y s t e m a t i c c o u n t i n g of a c a t e g o r y of o b -
jects;
(v) p l o t t i n g a s i m p l e o r a c c u m u l a t e d fre- 1.10 Phy sicochemical
quency distribution curve;
(vi) r e c o g n i t i o n of s h a p e s , a n a l y s i s of f o r m
microanalysis
factors; Micro-electrophoresis cell
(vii) a n a l y s i s of t e x t u r e s ; P R E P A R A T I O N O F A CELL BY C H O U C R O U N ' S
(viii) c l e a n i n g i m a g e s in o r d e r to e l i m i n a t e TECHNIQUE
b a c k g r o u n d noise, or objects which are T h e cell (Figure 1.100) c o n s i s t s of a t h i c k glass
n o t of i n t e r e s t ; p r o g r e s s i v e e r o s i o n of p l a t e ( a p p r o x . 6 m m ) w i t h p a r a l l e l faces (42 m m
objects; b y 32 m m ) . T h e u p p e r s u r f a c e of t h e t u b e h a s
(ix) m e a s u r e m e n t of l i n e a r c o n t o u r s o r p e r i - t w o U - s h a p e d g r o o v e s of c r o s s - s e c t i o n of a b o u t
m e t e r s of o b j e c t s ;
(x) i n d i c a t i o n of t h e f r e q u e n c y of a s e g m e n t Suspension of
red cells
in a c o m p o n e n t ;
(xi) m e a s u r e m e n t of t h e c u m u l a t e d d i s t r i b u -
t i o n of l e n g t h of c o r d s t r a v e r s e d o r i n t e r -
cepted;
(xii) c o r r e l a t i o n of t h e c o v a r i a n c e of t w o
p o i n t s o r of t w o c o m p o n e n t s ;
(xiii) a n a l y s i s of s u r f a c e s in c o n t a c t o r o v e r l a p -
ping;
a n a l y s i s of t h e v i d e o s i g n a l , in f r e q u e n c y
V
(xiv)

(xv)
or variance;
c o n t r a c t i o n a n d d i l a t a t i o n of i m a g e s a n d
L
a n a l y s i s of p r o x i m i t y ; F i g u r e 1.100. N. Choucroun's
electrophoresis cell
(xvi) a n a l y s i s of p o r o s i t y ;
Physicochemical microanalysis 91

10 m m , c o n n e c t e d b y a s m a l l r e c t a n g u l a r g r o o v e substances form a simple eutectic mixture,


( A B ) a p p r o x . 0.6 m m l o n g , 1 m m w i d e a n d m o l e c u l a r c o m p o u n d s or solid solutions,
a p p r o x . 0.15 m m d e e p . T h e p l a n e surfaces a n d t o g e t h e r w i t h v a r i o u s o t h e r a c c e s s o r i e s of lesser
t h e floor of t h e g r o o v e A B a r e g r o u n d a n d importance.
p o l i s h e d to o p t i c a l flatness. T o p r e p a r e t h e cell,
its u p p e r s u r f a c e is c o v e r e d w i t h a g l a s s slide HEATING MICROPLATES
w h i c h is s t u c k w i t h h o t paraffin. T h e s e a r e l o c a t e d i n s i d e a g l a z e d c h a m b e r of a n
T h e b a s e of t h e U t u b e s is filled w i t h h o t o b j e c t h o l d e r w h i c h c a n b e m a n i p u l a t e d from
v i s c o u s g e l a t i n (a s o l u t i o n of 4 0 g of p u r e g e l a t i n o u t s i d e , a n d c o n s i s t of a t h e r m o m e t e r o r p r o b e
in 100 m l of N / 1 K C l ) . T h e cell is a l l o w e d to cool w h i c h is c o n n e c t e d t o a m e a s u r i n g i n s t r u m e n t
u n t i l t h e g e l a t i n sets a n d m a k e s a c o m p l e t e a n d a h e a t e d r e s i s t o r w h i c h is c o n n e c t e d to a n
separation between the compartments. C o m - a d j u s t a b l e c u r r e n t s u p p l y . T h e single w i n d o w
p a r t m e n t s a n d F a r e filled w i t h t h e s o l u t i o n s t a g e s h o u l d n o t b e h e a t e d to a b o v e 3 0 0 C to
u n d e r test. T h e c o m p a r t m e n t s a r e t h e n s e a l e d p r e v e n t d a m a g e to t h e o b j e c t i v e . T o a t t a i n
w i t h h o t paraffin w h i c h is p o u r e d o n t h e t o p of higher temperatures, one must have a double
t h e cell. window stage, with an evacuated gap between
T h e c o m p a r t m e n t s C a n d D a r e filled w i t h t h e t w o g l a s s w a l l s (see Figure 1.77).
potassium chloride solution a n d the electrodes
are then introduced into the solution.
T h e cell is c o m p l e t e l y s e a l e d a n d a i r - t i g h t ,
t h u s e n s u r i n g safety a n d sterility d u r i n g h a n d -
ling a n d e x p e r i m e n t s . T h e thin glass cover
e n a b l e s o b s e r v a t i o n s to b e m a d e u n d e r h i g h
magnification, even with a n immersion objec-
tive.
T h e p r i n c i p l e of t h i s cell is to c o n c e n t r a t e t h e
m a j o r p a r t of t h e e n e r g y of t h e e l e c t r i c field in
t h e m e d i a , w h i c h a r e s u b j e c t e d to e l e c -
t r o p h o r e s i s , in s u c h a m a n n e r t h a t in t h e w e a k
field w h i c h is p r e s e n t n e a r t h e e l e c t r o d e s , t h e
+
H a n d O H ~ i o n s f o r m e d will m i g r a t e so slowly
that they do not reach the observed solution
d u r i n g t h e t i m e of t h e e x p e r i m e n t .
I n p r a c t i c e t h e e l e c t r i c field in t h i s cell is
c o n c e n t r a t e d e n t i r e l y in t h e r e g i o n A B for t w o
reasons:
F i g u r e 1.101. A mirror objective with
(1) T h e r a t i o b e t w e e n t h e c r o s s - s e c t i o n a l a r e a a large working distance, for
of t h e c h a m b e r A B a n d t h e c r o s s - s e c t i o n a l examining objects at high temperature
(Reichert, according to Nomarski).
a r e a of t h e U - g r o o v e s is s m a l l .
Magnification X40, numerical
(2) T h e c o n d u c t i v i t y of t h e g e l a t i n a n d p o t a s - aperture 0.52, working distance 8 mm
s i u m c h l o r i d e s o l u t i o n , a t t h e e l e c t r o d e s , is (Reichert)
h i g h c o m p a r e d to t h e c o n d u c t i v i t y of t h e
REFRIGERATED MICROSTAGES
solution u n d e r observation. (This a p p a r a t u s
By analogy with the accessory described above,
is t h u s m o s t effective w h e n t h e s u s p e n s i o n s
t h e s e h a v e a c o o l i n g d e v i c e , e i t h e r in t h e f o r m of
u n d e r observation h a v e a low conductivity.)
a P e l l e t i e r t h e r m o c o u p l e , o r a c i r c u i t in w h i c h
e x p a n d e d c a r b o n d i o x i d e g a s is c i r c u l a t e d . A n
a u x i l i a r y h e a t i n g d e v i c e p r o v i d e s for r a p i d r e -
Thermal m i c r o m e t h o d s
h e a t i n g between successive observations. T h e
T h e r m a l micromethods are based on the princi- r a n g e of t e m p e r a t u r e s c o v e r e d is u s u a l l y from
ples e s t a b l i s h e d b y L . a n d A . K f l e r for - 5 0 t o + 8 0 C . S o m e of t h e s e t w o t y p e s of p l a t e s
microchemical analysis. T h e y require a micro- h a v e e l e c t r o n i c p r e p r o g r a m m i n g of t h e t e m p e r a -
scope, preferably a polarizing microscope, a n d a t u r e r i s e , c o n t r o l of t h e h e a t i n g r a t e a n d a u t o m a -
h e a t i n g p l a t e in o r d e r to e s t a b l i s h w h e t h e r t w o tic r e c o r d i n g (see Figure 1.77).
92 Instruments and techniques

MICROSUBLIMATION BLOCK a l i q u i d of k n o w n r e f r a c t i v e i n d e x , it b e c o m e s
T h i s is a n a i r - t i g h t c h a m b e r w i t h a w i n d o w i n v i s i b l e if t h e r e f r a c t i v e i n d i c e s of t h e t w o
i n s i d e , in w h i c h it is p o s s i b l e to p r o d u c e a substances are equal at the standard tempera-
p a r t i a l v a c u u m , if n e c e s s a r y . T h e c h a m b e r is t u r e of 2 0 C . I f t h e r e is a s l i g h t difference in t h e
introduced into a heating microstage. refractive indices, the S c h r d e r - v a n d e r K o l k
fringe m e t h o d is u s e d t o e s t a b l i s h w h e t h e r t h e
OBJECTIVE PROTECTOR r e f r a c t i v e i n d e x of t h e solid is less o r g r e a t e r
I t is s o m e t i m e s n e c e s s a r y t o o p e r a t e w i t h o u t a t h a n t h a t of t h e m e d i u m in w h i c h it is i m m e r s e d .
window. Corrosive or o p a q u e v a p o u r might T h i s is h o w t h e m e t h o d w o r k s : b r i g h t field
d e p o s i t o n t h e front l e n s of a n o b j e c t i v e . T o i l l u m i n a t i o n is u s e d a n d h a l f t h e a p e r t u r e d i a -
protect the lens, we r e c o m m e n d that a small p h r a g m of t h e c o n d e n s e r is m a s k e d . T h e i m a g e
cover-slip be m a d e to a d h e r e to the objective is t h e n b r o u g h t s l i g h t l y o u t of focus, first u p a n d
w i t h a d r o p of oil o r g l y c e r i n . T h e c o v e r - s l i p is t h e n d o w n , a n d t h e d i s p l a c e m e n t of t h e first
r e m o v e d after u s e . b l a c k diffraction fringe is carefully o b s e r v e d o r
t h a t of t h e b r i g h t fringe w h i c h is l o c a t e d b e -
t w e e n t h i s a n d t h e o b j e c t . Figure 1.30 ( p . 2 4 )
Microrefractometry
shows how the observations are interpreted.
F o r m i c r o c h e m i s t r y o n e h a s t o m e a s u r e refrac-
tive i n d i c e s (Tables 1.2 a n d 1.3). T h e s e r e a d i n g s VARIATIONS DUE TO TEMPERATURE
h a v e t o b e m a d e o n v e r y m i n u t e a m o u n t s of T h e t e m p e r a t u r e coefficient of t h e refractive
s u b s t a n c e s , l i q u i d o r solid, a n d so t w o sets of i n d e x d e p e n d s o n t h e n a t u r e of t h e s u b s t a n c e ;
r e f e r e n c e o b j e c t s , s o l i d s a n d l i q u i d s , h a v e to b e g l a s s , for e x a m p l e , h a s a coefficient of 0 . 0 0 0 0 0 1
m a d e . W h e n t h e u n k n o w n o b j e c t is i m m e r s e d in p e r C ; o r g a n i c c o m p o u n d s h a v e coefficients

T a b l e 1.2 Kodak standard liquids for the determination of refractive indices

Standard liquid [ n D] 2 0 C Standard liquid [ n D] 20 C Standard liquid [ D] 2 0 C

Methanol 1.3288 Trimethylene chloride 1.4476 Methyl iodide 1.5310


Water 1.330 Cyclohexanone 1.4507 Ethylene bromide 1.5383
Acetone 1.3592 Cyclohexanol 1.4678 o-Nitrotoluene 1.5466
Ethyl acetate 1.3727 Diethanolamine 1.4782 Nitrobenzene 1.5526
rc-Hexane 1.3755 Triethanolamine 1.4853 Tri-o-cresylphosphate 1.5532
w-Heptane 1.3872 />-Cumene 1.4908 Bromobenzene 1.5602
w-Butanol 1.3991 i-Tetrachlorethane 1.4943 o-Toluidine 1.5725
w-Butyl chloride 1.4023 Toluene 1.4957 Aniline 1.5864
1,4-Dioxane 1.4222 Benzene 1.5017 Bromoform 1.5973
Methylcyclohexane 1.4235 Ethyl iodide 1.5138 o-Iodotoluene 1.6905
Ethyleneglycol 1.4318 Anisole 1.5178 Quinaldine 1.6120
Ethyl citrate 1.4434 Trimethylene bromide 1.5238 Iodobenzene 1.6205
Ethylene dichloride 1.4453 Chlorobenzene 1.5250

T a b l e 1.3 Isotropic crystals for the determination of refractive indices

Isotropic crystal [ D] 20 C Isotropic crystal [ n D] 20 C Isotropic crystal [D]20C

Sodium fluoride 1.326 Sodium chlorate 1.515 Potassium iodide 1.667


Potassium fluosilicate 1.339 Sodium chloride 1.544 Ammonium chlorostannate 1.678
Potassium fluoride 1.352 Rubidium bromide 1.553 Caesium bromide 1.698
Ammonium fluosilicate 1.370 Potassium bromide 1.559 Ammonium iodide 1.703
Potassium cyanide 1.410 Chromium sulphate 1.564 Ammonium bromide 1.711
Sodium alum 1.430 Barium nitrate 1.571 Magnesium oxide 1.736
Potassium alum 1.456 Strontium nitrate 1.586 Arsenic trioxide 1.755
Ammonium alum 1.459 Sodium bromate 1.617 Sodium iodide 1.775
Potassium chromium alum 1.481 Ammonium chloride 1.640 Lead nitrate 1.782
Ferriammonium alum 1.485 Sodium bromide 1.641 Caesium iodide 1.788
Potassium chloride 1.490 Caesium chloride 1.642 Potassium chloroplatinate 1.825
Rubidium chloride 1.494 Rubidium iodide 1.647
Sodium uranyl acetate 1.504 Potassium chlorostannate 1.657
Physicochemical microanalysis 93

4 2

4 2

Figure 1.102. Refractive index of a water-glycerin mixture.


The ordinates represent refractive indices, the abscissae the
glycerin content in the mixture (%)

r a n g i n g from 0.0002 to 0.0009 p e r C . I t is


a d v i s a b l e , t h e r e f o r e , t o k e e p t o a t e m p e r a t u r e of
a b o u t 2 0 C w h e n c o n d u c t i n g r e a d i n g s in y e l l o w
s o d i u m light, a n d to m a k e a careful r e c o r d of t h e
t e m p e r a t u r e , n' = 0 . 0 0 0 4 / is a n a v e r a g e
correction formula.

O P T I C A L M O D I F I C A T I O N S FOR E X A M I N A T I O N S
S o m e a u t h o r s prefer to t a k e r e a d i n g s u n d e r F i g u r e 1.103. Principle of various techniques of
autoradiography. 1, supporting slide; 2, histological
p h a s e contrast with the object presenting a section; 3, radio-sensitive emulsion; 4, cover-slip
d a r k e r p o s i t i v e field p h a s e , if its i n d e x is g r e a t e r (Policard, Bessis and Locquin)
t h a n t h a t of t h e m e d i u m o r , c o n v e r s e l y , l i g h t e r if
its i n d e x is l o w e r . w i t h t h e e l e c t r o n m i c r o s c o p e . T h e m o v e m e n t s of
I f i n t e r f e r e n c e c o n t r a s t is a v a i l a b l e , t h i s is t h e l a b e l l e d c o m p o u n d a t t h e tissue a n d in-
m u c h t h e b e t t e r m e t h o d b e c a u s e it p r o v i d e s t r a c e l l u l a r levels, h o w e v e r , a r e fields in w h i c h
m e a n s of e x a c t c a l c u l a t i o n of t h e refractive t h e m e t h o d excels, e v e n b e t t e r t h a n t h e s t a t i c
i n d e x , e v e n if it is o n l y slightly different from d e t e r m i n a t i o n of t h e l o c a t i o n s of c o m p o u n d s . I t
t h a t of t h e r e f e r e n c e l i q u i d o r c r y s t a l , b y is w i t h o u t e q u a l in this a p p l i c a t i o n . T h e
m e a s u r i n g t h e differences in s t e p b e t w e e n t h e r a d i o i s o t o p e s u s e d for t h i s p u r p o s e h a v e half-
o b j e c t a n d t h e m e d i u m (cf. p p . 3 8 - 4 0 ) . lives of a t m o s t a few h o u r s a n d e m i t m a i n l y oc-
W h e n o p e r a t i n g in a l i q u i d of sufficiently a n d - r a d i a t i o n (Figure 1.103).
large volume, the Nichols refractometer can be
u s e d . A d o u b l e p r i s m a c c o m m o d a t e s t h e d r o p of CONTACT AUTORADIOGRAPHY

l i q u i d to b e m e a s u r e d ; t h e refractive i n d e x is T h i s is t h e s i m p l e r m e t h o d b u t d o e s n o t define
obtained by reading the interval between two t h e l o c a t i o n of t h e r a d i o - l a b e l l e d s u b s t a n c e ,
lines after c a l i b r a t i o n . e x c e p t a t a t i s s u e level. D r y s e c t i o n s o r s e c t i o n s
Figure 1.102 s h o w s t h e refractive i n d e x of a in w a t e r a r e s p r e a d o u t o n p h o t o g r a p h i c e m u l -
waterglycerin m i x t u r e . s i o n . T h e s e c t i o n follows t h e e m u l s i o n t h r o u g h -
o u t all t h e o p e r a t i o n s . T h i s m e t h o d is g r e a t l y
s u p e r i o r to t h e t e c h n i q u e in w h i c h slides a r e
Autoradiography
p l a c e d in c o n t a c t w i t h e m u l s i o n s , b e c a u s e of t h e
T h i s m e t h o d h a s b e e n k n o w n s i n c e 1924 a n d it difficulty in t h i s l a t t e r c a s e of d e f i n i n g p o s i t i o n s .
b e c a m e p o p u l a r w i t h t h e a p p l i c a t i o n of r a d i o a c - S u i t a b l e p r o c e s s e s m u s t n o t p r o d u c e c h a n g e s in
tive l a b e l l e d c o m p o u n d s to b i o l o g y . T h e a c c u r a - s e c t i o n s o r s t a i n t h e m b y excessive p h o t o g r a p h i c
cy w i t h w h i c h c o m p o u n d s a r e l o c a t e d is s u c h emulsion. T h e m e t h o d can be improved by
that some preparations have been investigated s a n d w i c h i n g the section between two emulsions.
94 Instruments and techniques

IMPREGNATION AUTORADIOGRAPHY v a c u u m , o n e c a n u s e e v e n softer r a d i a t i o n ( =


T h i s gives m u c h m o r e p r e c i s e r e s u l t s . T h e sec- 2 - 5 n m ) . T h e best performances obtained en-
t i o n is i m p r e g n a t e d w i t h a p h o t o g r a p h i c e m u l - a b l e p l a t e s to b e e x a m i n e d w i t h m a g n i f i c a t i o n s
sion w h i c h m e l t s a t a b o u t 3 7 C . T h e e m u l s i o n s of t h e o r d e r of 8 0 0 . A t o m s of h e a v y e l e m e n t s a r e
c a n t h u s b e m e l t e d a t t h e surface a l o n e , w h i l e a r e a d i l y identified o n t h e s e p l a t e s , b u t w i t h o u t
l a y e r of c o l l o d i o n c a n b e i n t e r p o s e d b e t w e e n t h e i n d i c a t i o n of w h a t c o m b i n a t i o n s t h e y a r e in.
t w o to a v o i d diffusion effects. O n e c o m p a r e s t h e m w i t h a scale c o n s t i t u t e d b y
n i t r o c e l l u l o s e s e c t i o n s to d e t e r m i n e t h e o v e r a l l
PEELED FILM A U T O R A D I O G R A P H Y d r y w e i g h t of a s e c t i o n , b u t this c a n b e d o n e
T h i s m e t h o d c o n s i s t s in c o v e r i n g t h e c o l l o d i o n - m o r e cheaply by interference contrast micros-
c o v e r e d s e c t i o n w i t h a l a y e r of a n e m u l s i o n copy.
w h i c h c a n b e s t r i p p e d off its b a c k i n g .
All t h e s e o p e r a t i o n s s h o u l d b e c o n d u c t e d SHADOW MICRORADIOGRAPHY OR THE
u n d e r p h o t o g r a p h i c 'safe-light'. E x p o s u r e t i m e s MAGNIFICATION METHOD
m a y v a r y from a few h o u r s to s e v e r a l m o n t h s . T h i s m e t h o d has the s a m e limitations as the
O n e c o u n t s t h e g r a i n s of silver after d e v e l o p - previous method, and has an additional draw-
m e n t . C e r t a i n d y e s fade o r d i s a p p e a r in t h e b a c k in t h a t it is difficult to o b t a i n s u i t a b l e
d e v e l o p i n g o r fixing b a t h s if s t a i n i n g is c a r r i e d i n t e n s i t i e s to p r o v i d e e x p o s u r e t i m e s w h i c h a r e
o u t before d e v e l o p m e n t . s h o r t e n o u g h , a n d still h a v e a s m a l l e n o u g h
s o u r c e of r a d i a t i o n . By u s i n g m o n o c h r o m a t i c
X - r a y s , it h a s b e e n p o s s i b l e to u s e t h e s e last t w o
Historadiography m e t h o d s to d e t e c t c e r t a i n c h e m i c a l e l e m e n t s
from d i s c o n t i n u i t i e s in a b s o r p t i o n c u r v e s .
T h e r e is a n a c c u r a t e l y defined r e l a t i p n b e t w e e n T h e u s e of a s y n c h r o t r o n r a y p r o d u c e d b y
a b s o r p t i o n of X - r a y s b y a s t r u c t u r e a n d t h e collision a n n u l i p e r m i t s o n e to u s e m a n y k i n d s of
a t o m i c n u m b e r s of t h e e l e m e n t s p r e s e n t ; h i s t o - X - r a y s in m i c r o s c o p y . T h i s i n s t r u m e n t d e l i v e r s
r a d i o g r a p h y is b a s e d o n t h i s . S i n c e t h e a d v e n t of a c o n t i n u o u s i n f r a r e d r a y to t h e X - r a y s w i t h
e l e c t r o n i c p r o b e s , t h e m e t h o d h a s lost s o m e of c o n s i d e r a b l e i n t e n s i t y . T h i s r a y is p u l s e d a n d
its o r i g i n a l p o p u l a r i t y , b e c a u s e t h e s c a n n i n g polarized. It decreases the normal exposure time
electron microscope, when suitably equipped, for m i c r o r a d i o g r a p h y b y a n o r d e r of a t h o u s a n d .
c a n yield t h e s a m e i n f o r m a t i o n (cf. p . 9 5 ) .
X - r a y s c a n b e u s e d in t h r e e w a y s : t r u e m i c r o s -
copy, contact microradiography and micro- Spodography
radiography by s h a d o w or by projection.
M i c r o - i n c i n e r a t i o n is a m e t h o d of h i s t o s p o -
X-RAY M I C R O S C O P Y d o g r a p h y w h i c h s h o w s t h e m i n e r a l s k e l e t o n of
T h i s u s e s a s o u r c e , as close a s p o s s i b l e to a p o i n t a n o b j e c t after h e a t i n g a n d v o l a t i l i z i n g all its
s o u r c e , focused b y t w o c r y s t a l l a t t i c e s w h i c h o r g a n i c c o n s t i t u e n t s . T h e r e s u l t is called a
have been mechanically curved and oriented at s p o d o g r a m . R a s p a i l in 1833 a p p l i e d this m e t h o d
right angles. T h e b e a m should be at glancing t o p l a n t cells, b u t a c e n t u r y p a s s e d b y before t h e
i n c i d e n c e a n d t h e e n e r g y b e v e r y low. T h e w o r k of A . P o l i c a r d m a d e t h e m e t h o d w o r t h y of
r e s u l t s o b t a i n e d b y this m e t h o d a r e d e c e p t i v e , r e f e r e n c e . T h e s p o d o g r a m gives a c o m p l e t e r e p -
t h e r e f o r e , a n d n o e q u i p m e n t of t h i s t y p e h a s r e s e n t a t i o n of t h e o v e r a l l d i s t r i b u t i o n of m i n e r -
been found commercially viable. A second a l s in a cell. T h e m o s t d e l i c a t e s t r u c t u r e s a r e
m e t h o d w h i c h is m u c h m o r e p r o m i s i n g is n o w p r e s e r v e d to s u c h a n e x t e n t t h a t t h e y c a n b e
b e i n g i n v e s t i g a t e d b y o n e of t h e a u t h o r s . e x a m i n e d in t h e e l e c t r o n m i c r o s c o p e .
F i x a t i v e s w h i c h c o n t a i n o r dissolve i n o r g a n i c
CONTACT MICRORADIOGRAPHY c o m p o u n d s c a n n o t b e u s e d for fixation. T h e
T h i s m e t h o d is l i m i t e d b y t h e fineness of t h e s e c t i o n h a s to b e p r o d u c e d b y freezing, o r in
s o u r c e , t h e g r a i n of t h e e m u l s i o n a n d t h e resolv- paraffin. C o l l o d i o n is n o t to b e u s e d b e c a u s e of
i n g p o w e r of t h e m i c r o s c o p e , since t h e p l a t e h a s its e x p l o s i v e c h a r a c t e r w h i c h w o u l d d i s i n t e g r a t e
t o b e e x a m i n e d w i t h t h e light m i c r o s c o p e . the sections w h e n they are heated. Sections are
O n l y v e r y soft r a d i a t i o n , of w a v e l e n g t h r a n g - m a d e a n d m o u n t e d n o r m a l l y , b u t u s i n g paraffin
i n g from 0.1 to 1 n m , e m i t t e d b y a low v o l t a g e i n s t e a d of w a t e r a n d w i t h slides m a d e from
s o u r c e c a n b e u s e d . If o n e o p e r a t e s in a h i g h r e f r a c t o r y g r e e n b a n d g l a s s . T h e slide is i n s e r t e d
Microanalysis by electron microscopy 95

in a c a l i b r a t e d q u a r t z t u b e w h i c h is s u r r o u n d e d weight or heavy elements. S o m e small objects


by a heating resistor a n d sheathed with asbestos c a n b e i n c i n e r a t e d in toto, e.g. p r o t o z o a , t h e
o r a P o l i c a r d f u r n a c e . T h e t e m p e r a t u r e is r a i s e d s p o r a n g i a of fungi, filaments o r fibres.
g r a d u a l l y to 70 C in 10 m i n , t h e n left a t t h i s
t e m p e r a t u r e for 5 m i n to p r e v e n t s h r i n k a g e of
t h e c o l l a g e n , before b e i n g h e a t e d to 150 C o v e r
a p e r i o d of 15 m i n , to c o n v e r t all t h e o r g a n i c
m a t t e r to a s h . I f t h e o p e r a t i o n is s t o p p e d a t t h i s 1.11 Microanalysis by
s t a g e , t h e r e s u l t is a b l a c k a n t h r a c o g r a m . By electron microscopy
r a i s i n g t h e t e m p e r a t u r e still f u r t h e r to 5 0 0 C
a n d l e a v i n g t h e slide a t t h i s t e m p e r a t u r e , o r a t
T h e s c a n n i n g electron microscope
6 0 0 C for 20 m i n , a w h i t e o r r e d d i s h s p o d o g r a m
is o b t a i n e d . T h i s is t h e u l t i m a t e e x t e n s i o n to t h e infinitesi-
S p o d o g r a m s are then m o u n t e d as quickly m a l l y s m a l l of t h e s t e r e o s c o p i c m i c r o s c o p e o r
a s p o s s i b l e , d r y , b e t w e e n slide a n d c o v e r - s l i p , b i n o c u l a r m a g n i f i e r . I n s t e a d of e x a m i n i n g o b -
w i t h a s p a c e r of a d e q u a t e t h i c k n e s s i n t e r p o s e d j e c t s b y reflected p h o t o n s , i n f o r m a t i o n a b o u t t h e
to avoid crushing: s p o d o g r a m s are hygroscopic s u r f a c e is d e r i v e d from s c a n n i n g t h e surface w i t h
a n d fragile. T h e y a r e e x a m i n e d in b r i g h t field, a fine b e a m of e l e c t r o n s . U n l i k e t h e light m i c r o -
o r b e t t e r still in i n c i d e n t , i l l u m i n a t i o n , o r in s c o p e , t h e p a r t i c l e s w h i c h a r e u s e d to form t h e
d a r k field, p h a s e c o n t r a s t o r i n t e r f e r e n c e c o n - i m a g e o r a n a l y s e t h e c o m p o s i t i o n of t h e i m a g e
trast. a r e n o t a l w a y s of t h e s a m e t y p e (Figure 1.104).
S p o d o g r a m s c a n b e a n a l y s e d in t h e s c a n n i n g Figure 1.105 s h o w s a c r o s s - s e c t i o n of this
e l e c t r o n m i c r o s c o p e to identify a t o m s of m e d i u m e q u i p m e n t , b u t not illustrating the supply unit,

Figure 1.104. Camca scanning electron microscope


96 Instruments and techniques

Figure 1.105. Elmiskop ST 100F (Siemens) scanning microscope. Section to the left, diagrammatic representation on the right.
Left1, electron gun assembly; 2, diaphragm chamber; 3, deflector coils; 4, object chamber; 5, auxiliary sight; 6, waveguide.
Right1, filament; 2, Wehnelt; 3 and 6, diaphragms; 4, coil; 5 and 7, lenses; 8, object; SE back-scattered electrons; hv
back-scattered X-rays; 9, diaphragm; 10, detector plate; 11, waveguide; 12, diaphragm; 13, detector plate; 14, synchronized
deflector plates (Siemens)

the image synthesizer or the measuring instru- a t e d . T h e b a c k - s c a t t e r e d e l e c t r o n s a r e reflected,


m e n t s w h i c h a r e c o n n e c t e d to t h e d e t e c t o r s . w i t h o u t e n e r g y loss a t t h e c r y s t a l p l a n e s of t h e
W h e n a b e a m of e l e c t r o n s i m p i n g e s o n a n s u b s t a n c e ; o t h e r s suffer e n e r g y losses w h i c h a r e
o b j e c t in a v a c u u m , t h e p a r t i c l e s m a y b e a f u n c t i o n of t h e n a t u r e of t h e s u b s t a n c e e n c o u n -
a b s o r b e d , t r a n s m i t t e d or their frequency t e r e d , a n d t h i s effect c a n b e e x p l o i t e d for a n a l y -
c h a n g e d in v a r i o u s w a y s . T h e t r a n s m i t t e d elec- sis.
t r o n s , after p a r t i a l a b s o r p t i o n b y t h e o b j e c t , a r e T h e p r i n c i p l e of s c a n n i n g e l e c t r o n m i c r o s c o p y
u s e d to form a t r a n s m i t t e d i m a g e , a s is d o n e ( S E M ) is s i m i l a r to t h a t of television, in t h a t t h e
w i t h p h o t o n s in b r i g h t field light m i c r o s c o p y . s u r f a c e of a f l u o r e s c e n t s c r e e n r e p r o d u c e s p o i n t
E l e c t r o n s of t h e A u g e r , b a c k - s c a t t e r e d o r s e c o n - by point the information obtained, by secondary
d a r y t y p e s , c a n b e d e t e c t e d i n d e p e n d e n t l y , to electrons, back-scattered electrons or absorbed
f o r m i m a g e s o r to p r o v i d e a s p e c t r o - a n a l y t i c a l electronsthe information being obtained by
c u r v e . T h e s a m e h o l d s t r u e of t h e c a t h o d e s u i t a b l e d e t e c t o r s (Figure 1.105).
luminescence and the X-rays which are gener- T h e secondary electrons, which are displaced
Microanalysis by electron microscopy 97

from their orbitals by incident b o m b a r d m e n t , freezing of t h e s p e c i m e n , in situ, p r o v i d e s a


a r e n o t c h a r a c t e r i s t i c of a s p e c i m e n . T h e y c a n m e t h o d of o b s e r v i n g it, w i t h o u t m e t a l - c o a t i n g ,
b e u s e d to f o r m a c o m p l e t e , n o n - s e l e c t i v e i m a g e provided that the emitted electrons arc control-
of a g i v e n c o n s t i t u e n t . If a m e t a l l i c c o a t i n g is l e d , t o b e v e r y n e a r l y e q u a l to t h e i n c i d e n t
applied to the s u b s t a n c e , the emission can be e l e c t r o n s , t o a v o i d a c c u m u l a t i o n of electric
i m p r o v e d by b a l a n c i n g the surface polarization c h a r g e . Biological objects are n o n - c o n d u c t o r s ,
p o t e n t i a l s a n d c o m p e n s a t i n g for t h e low a t o m i c h e n c e a n y d e p a r t u r e from t h i s s t a t e i m p e d e s
w e i g h t s of s o m e p o i n t s o n t h e s p e c i m e n . e m i s s i o n b e c a u s e of a c c u m u l a t i o n of c h a r g e s .
T h e c h a r a c t e r i s t i c s of t h e s e c o n d a r y e m i s s i o n W h e n a n a t o m is s t i m u l a t e d b y i n c i d e n t
d e t e r m i n e t h e c o n t r a s t in t h e i m a g e . T h e m a i n e l e c t r o n s , a n e l e c t r o n in a m e d i u m s t a t e is r a i s e d
o n e is t h e a n g l e of i n c i d e n c e of t h e b e a m , w h i c h t o a h i g h e r level. W h e n it falls b a c k , it e m i t s
c a n give a n i m p r e s s i o n of s u r f a c e relief, s i m i l a r either X - r a y fluorescence or A u g e r electrons
to t h a t given by the stereoscopic light micro- w h i c h h a v e b e e n d e t a c h e d from t h e s u r f a c e
scope. T h e resolving power d e p e n d s on the level. T h e g e n e r a t i o n of X - r a y s is m o r e p r o b a b l e
fineness of t h e s c a n n i n g b e a m a n d its h i g h t h a n A u g e r e l e c t r o n s in t h e c a s e of h e a v y ele-
d e g r e e of c o l l i m a t i o n ; this p r o v i d e s a v e r y g r e a t m e n t s , a n d vice v e r s a . T h e t w o t y p e s of r a d i a -
d e p t h of field, g r e a t e r t h a n t h a t of t h e l i g h t t i o n a r e c h a r a c t e r i s t i c of t h e a t o m s w h i c h g e n e r -
m i c r o s c o p e a t t h e s a m e m a g n i f i c a t i o n a n d giv- ate them.
i n g a r e s o l v i n g p o w e r w h i c h is i n c o m p a r a b l y X - r a y s can be detected by dispersion spec-
higher. T h e second component depends u p o n trometry by wavelength a n d by energy. F r o m
t h e a t o m i c w e i g h t s of t h e m o l e c u l e s w h i c h a r e b e r y l l i u m t o u r a n i u m , e l e m e n t a r y a n a l y s i s is
e n c o u n t e r e d . T h e images yielded by light a n d conducted either with an X-ray and Auger
heavy elements are thus strongly differentiated. image, or with an X-ray and Auger spectrum.
T h e i m a g e of t h e e l e c t r o n s a b s o r b e d is a p p r o x - S o m e i n s t r u m e n t s p r o v i d e m e a n s for s u p e r i m -
i m a t e l y c o m p l e m e n t a r y to t h a t of s e c o n d a r y p o s i n g a n i m a g e a n d a s p e c t r a l line, t h u s facili-
p l u s t h e b a c k - s c a t t e r e d e l e c t r o n s (Figures 1.106- t a t i n g i n t e r p r e t a t i o n . W e c a n briefly list s o m e
1.108). o t h e r possibilities w i t h o u t going into details:
diffraction d i a g r a m s ; e x a m i n a t i o n of fluoresc-
O B S E R V A T I O N S IN FROZEN SPECIMENS e n c e effects; p o t e n t i a l c o n t r a s t s t u d i e s .
F r a g i l e b i o l o g i c a l s p e c i m e n s a r e l i a b l e to b e M o s t i n s t r u m e n t s c a n b e s u p p l i e d in different
d a m a g e d when they are metal-coated. Deep d e s i g n s , f r o m t h e s i m p l e s t w h i c h is c o n f i n e d t o

Figure 1.106. Comparison between images obtained in the light microscope (transmitted light) on the left, and the scanning
electron microscope (right) showing two similar diatoms, Tnceratium morlandi Grev. (left) and Triceratium formosum Brightwell
(right); the magnification is about X1300. The image obtained in the scanning electron microscope is very similar to those
obtained at lower magnification in the stereoscopic light microscope (Cryptogamy Laboratory, National Natural History
Museum, Paris) (Documents P. Fuseyleft; M. Ricardright)
98 Instruments and techniques

F i g u r e 1.107. A tetrad of basidiospores of Chlorophyllum


molybdites (May ex. Fries) Mas. in the scanning electron
microscope. It can be seen that at this high magnification of
approximately X3000 there is a great depth of field and
perfect reproduction of the relief (Photomicrograph from the
Geology Laboratory, Jacqueline Perreau, Cryptogamy
Laboratory, National Natural History Museum, Paris)

Figure 1.108. Basidiospores of Rozites caperata (Pers. ex Fr.) Karst (left) and of Russuta clariana Heim (right). The
magnification is approximately X5000 (From same source as Figure 1.107)

morphological examination using secondary a l s o a p p l y a d r o p of a q u e o u s o r e t h a n o l i c s u s -


e l e c t r o n s , to a m e d i u m g r a d e i n s t r u m e n t w h i c h pension which contains the objects, which
gives a n o v e r a l l i m p r e s s i o n of t h e c o m p o s i t i o n a d h e r e s p o n t a n e o u s l y to t h e slide after e v a p o r a -
u s i n g b a c k - s c a t t e r e d e l e c t r o n s , to a m o r e s o p h - t i o n of t h e v e h i c l e .
isticated version which provides elementary
analysis by X-rays a n d Auger electrons, and APPLICATION OF METAL COATING T O
finally to t h e c o m p r e h e n s i v e m o d e l s u s e d in SPECIMENS
physics laboratories. A l a y e r less t h a n l O n m of g o l d , p l a t i n u m ,
g e r m a n i u m , o r a n alloy of g o l d a n d p a l l a d i u m o r
M O U N T I N G T H E SPECIMENS g o l d a n d c o p p e r is d e p o s i t e d . T h e c o a t i n g c a n
T h i s is d o n e o n a m e t a l slide, u s u a l l y 8 - 1 0 m m b e a p p l i e d d i r e c t l y , o r after first c o a t i n g t h e
c o p p e r b l o c k s . T h e y a r e selected a n d o r i e n t a t e d s p e c i m e n w i t h c a r b o n , if t h e s h a p e is to b e
m a n u a l l y o r w i t h t h e a i d of a m i c r o d i s s e c t o r , r e t a i n e d , w h i l e a v o i d i n g s h r i n k a g e effects. T h e
a n d t h e o b j e c t s a r e t h e n g l u e d to t h e s t a g e w i t h r e s o l v i n g p o w e r is 2 0 - 8 n m , in t h e o r y , after
adhesive or double sided adhesive tape. O n e can m e t a l l i z a t i o n (Figure 1.109).
Microanalysis by electron microscopy 99

CONSTRAINTS AND LIMITATIONS


T h e main constraints are:

(a) o p e r a t i o n s m u s t a l w a y s b e c o n d u c t e d u n d e r
vacuum;
( b ) o b s e r v a t i o n is confined to t h e surface b y
r e f l e c t i o n t h e r e is n o p e n e t r a t i o n ;
(c) o n e c a n n o t s t a i n o r differentially i m p r e g -
n a t e t h e t i s s u e s ; t h e y a r e s e n s i t i v e to i m -
p u r i t i e s o r r e s i d u e s left from p r e c e d i n g o p -
e r a t i o n s ; it is difficult to r e m o v e s o l v e n t s ,
fixing a g e n t s o r p r e s e r v a t i v e s t h e s p e c i -
m e n h a s to b e d r i e d w i t h o u t d e f o r m a t i o n o r
h a s to b e o b s e r v e d in t h e frozen s t a t e ;
(d) s o m e s p e c i m e n s a r e n a t u r a l l y fragile;
Figure 1.109. Shadowing and carbon coating (e) s o m e fragile s p e c i m e n s a r e c h a r r e d ;
techniques. The substance to be used for coating is (f) it is difficult to s t r i p m e t a l c o a t i n g s w i t h
vaporized in a bell jar under high vacuum from a
a q u a regia, a n d to recover the specimens.
heated filament or in a heating chamber. Carbon is
coated by striking an arc between two graphite T h e l i m i t s a r e r e l a t e d to t h e r e s o l v i n g p o w e r
electrodes, with the specimen held at right angles to
a n d t h e w i d e g a p b e t w e e n this e q u i p m e n t a n d
the source
the transmission electron microscope.

DEMETALLIZATION
SPECIAL T E C H N I Q U E S
T h i s p r o c e d u r e is p r a c t i c a b l e o n l y in s p e c i m e n s
I f s p e c i m e n s a r e m o u n t e d o n a c o v e r g l a s s slide,
w h i c h h a v e b e e n m o u n t e d o n g l a s s slides w i t h
they can be examined, subject to certain precau-
a d h e s i v e . T h e following t e c h n i q u e is t h e o n e
t i o n s , in t h e l i g h t m i c r o s c o p e , before a n d after
used by J . T a u g o u r d e a u , G. Lachkar, E. M a -
m e t a l l i z i n g a n d e x a m i n a t i o n in t h e s c a n n i n g
s u r e a n d J . C . A u d r a n ( 1 9 7 3 ) . T h e c o v e r glass is
electron microscope. T h i s permits better com-
r e m o v e d after o b s e r v a t i o n , u s i n g a r a z o r b l a d e ,
p a r i s o n w i t h t h e u s u a l i m a g e s ( A u b r y , 1973).
a n d is t h e n p l a c e d in a w a t c h g l a s s ; 3 - 4 d r o p s of
Before m e t a l c o a t i n g s a r e a p p l i e d , o n e c a n u s e
a q u a r e g i a a r e a d d e d . W i t h i n less t h a n 2 m i n t h e
selective s t r i p p i n g t e c h n i q u e s , i n t r o d u c e d b y
t r a n s p a r e n c y of t h e s e c t i o n i n d i c a t e s t h e e n d of
L o c q u i n (1942) for l i g h t m i c r o s c o p y . P o l i s h e d
t h e d e m e t a l l i z a t i o n . O n e millilitre of distilled
s u r f a c e s a n d t h i n slices c a n also b e u s e d a n d
w a t e r is a d d e d a n d t h e s p e c i m e n is t r a n s f e r r e d
stripped.
t o a s e c o n d w a t c h g l a s s w h i c h c o n t a i n s distilled
w a t e r . T h e d r o p of a d h e s i v e b e c o m e s o p a q u e
FRACTURING ROCKS CONTAINING
MICROFOSSILS d u r i n g t h e w a s h i n g , b u t r e c o v e r s its t r a n s p a r e n -
W i t h the scanning electron microscope one can cy o n h e a t i n g to 6 0 C . T h e s p e c i m e n s a r e
o b s e r v e in situ o n t h e s u r f a c e s u c h microfossils a s m o u n t e d b e t w e e n slide a n d c o v e r glass in a
spores, pollens or architarchs, by observing the hygroscopic liquid. Ethanol should not be used
f r a c t u r e d face of t h e rock. U s u a l l y , w h e n t h e a s it m a k e s s p e c i m e n s o p a q u e .
fossils h a v e b e e n p r e s e r v e d in o r g a n i c m a t t e r , O n e c a n e x t r a c t t h e m e t a l from a s p e c i m e n
t h e y d o n o t a d h e r e to t h e r o c k a n d a r e visible in containing gold or a l u m i n i u m with mercury or
a k i n d of n i c h e ( L o c q u i n , 1977; 1978). m e r c u r y vapour, or with mercuric chloride. T h e
s p e c i m e n is w a s h e d w i t h distilled w a t e r before
GRINDING AND DIFFERENTIAL FLOTATION continuing the procedure.
T h e s t o n e is c r u s h e d to a p o w d e r w h i c h is
s u s p e n d e d in w a t e r . A j e t of a i r from a n a r r o w ADHESIVES
g l a s s t u b e c a u s e s b u b b l e s to rise to t h e s u r f a c e Leffingwell a n d H o d g k i n (1971) r e c o m m e n d
a n d concentrate organisms liberated by the P V C w h i c h is s o l u b l e in m e t h y l e t h y l k e t o n e ,
g r i n d i n g . T h e s e m a y b e c l a s s e d a c c o r d i n g to d i l u t e d to a s y r u p y c o n s i s t e n c y . After surface
t h e i r size o r d e n s i t y b y b l o w i n g m o r e o r less e v a p o r a t i o n h a s h a r d e n e d t h e d r o p a little, it is
strongly, a n d a d d i n g surface active agents (Loc- a p p l i e d a n d s p r e a d o n t h e b a s e in o r d e r to
q u i n , 1978; 1979; 1980). m o u n t the specimens which adhere under gentle
100 Instruments and techniques

p r e s s u r e . T h e glass c o v e r - s l i p is fixed to t h e w a v e l e n g t h of t h e i l l u m i n a t i n g light. T h e resolv-


c o p p e r b a s e w i t h a s o l u t i o n of a silver salt. i n g p o w e r of t h e light m i c r o s c o p e c a n b a r e l y g o
b e y o n d 0.2 in p r a c t i c e , w h e r e a s t h e e l e c t r o n
MICRODISSECTORS microscope can be almost one thousand times
T h e c h o i c e d e p e n d s o n t h e d i m e n s i o n s of t h e b e t t e r . T h i s p e r f o r m a n c e h a s b e e n a c h i e v e d as a
s p e c i m e n s ; o n e m a y use a m i c r o d i s s e c t o r w i t h a r e s u l t of p r o l o n g e d r e s e a r c h , a n d h a s led to t h e
p a n t o g r a p h s y s t e m for r e d u c t i o n , a m i c r o d i s s e c - d e v e l o p m e n t of a n i n s t r u m e n t w h i c h is c o m p l e x
tor with a d y n a m i c reduction system by viscous a n d e x p e n s i v e ; its m a i n d r a w b a c k for u s e in
b l o c k s , o r a m i c r o d i s s e c t o r w h i c h is a t t a c h e d to b i o l o g y is t h e n e c e s s i t y to o p e r a t e u n d e r h i g h
t h e o b j e c t i v e of t h e o b s e r v i n g m i c r o s c o p e . v a c u u m with specimens which are extremely
thin. Despite this limitation, the electron micro-
S T E R E O S C O P I C VIEWS s c o p e c a n c l a i m to h a v e p r o d u c e d in 50 y e a r s
T h r e e - d i m e n s i o n a l m o d e l s c a n b e b u i l t u p from m o r e i n f o r m a t i o n of t h e i n t i m a t e k n o w l e d g e of
scanning electron micrographs by taking photo- t h e cell a n d tissues t h a n t w o c e n t u r i e s of t h e
g r a p h s a t s e v e r a l a n g l e s of tilt. O n e m u s t m a r k a l i g h t m i c r o s c o p e (Figure 1.110).
p o i n t a t t h e c e n t r e of t h e field to m a t c h u p t h e
v i e w s of t h e s a m e field.

T h e transmission electron microscope


T h i s is t h e e x t e n s i o n of t h e t r a n s m i t t e d light
m i c r o s c o p e to t h e infinitesimally s m a l l , i.e. in
b r i g h t field o p e r a t i o n . I t s d e v e l o p m e n t c a m e
b e c a u s e of t h e necessity for p r o g r e s s i v e l y finer
a n a l y s i s of biological s t r u c t u r e s , b e y o n d t h e
limit imposed on resolving power by the

iFigure 1.111. Section through the Philips


E M 400 electron microscope. 1, seven
lenses; 2, ion pump; 3, plate camera;
Figure 1.110. Philips transmission electron microscope 4, vibration-proof suspension for the
(Philips, Eindhoven) instrument (Philips, Eindhoven)
Microanalysis by electron microscopy 101

Photographic Fluorescent
plate of screen
ground glass

Eyepiece - Eyepiece

Objective
Objective
Specimen Specimen

F i g u r e 1.112. Comparative diagrams of the


Condenser Condenser
beam path in the light microscope (left) and
electron microscope (right). In practice, most
S O U R C FE O electron microscopes are designed with the same
Light source TH'ltl^AV""" arrangement as inverted light microscopes
1
II elftrtrons

Figure 1.111 is a s e c t i o n i n d i a g r a m m a t i c f o r m PRECIPITATION OF DIFFUSIBLE SUBSTANCES


through a transmission electron microscope T h e s e substances can b e characterized cyto-
( T E M ) . T h e g e n e r a l d e s i g n is t h e r e v e r s e o f t h a t chemically, by microwaves or by ionic analysis
u s e d for t h e l i g h t t r a n s m i s s i o n m i c r o s c o p e ; t h e after t h e y h a v e b e e n p r e c i p i t a t e d . L c h l i (1975)
e l e c t r o n s o u r c e is a t t h e t o p of a c o l u m n w h i c h a n d L o c q u i n ( 1 9 7 8 ) r e c o m m e n d e d , for t h e d e -
c a r r i e s t h e following t r a i n of c o m p o n e n t s : a t e c t i o n of t h e i o n s i n d i c a t e d , t h e following p r e -
b e a m c o n d e n s e r (single s t a g e o r t w o s t a g e ) , t h e c i p i t a t i n g a g e n t s : Cl~, silver a c e t a t e , silver l a c -
+ 2 1
specimen, a n objective, a n intermediate g u n , a t a t e ; N a , p o t a s s i u m p y r o a n t i m o n a t e ; Ca " ",
final g u n ( c o r r e s p o n d i n g t o t h e e y e p i e c e of t h e potassium oxalate, a m m o n i u m oxalate; P O l " ,
l i g h t m i c r o s c o p e ) , a fluorescent s c r e e n o n w h i c h lead acetate; A m " , silicotungstic acid; A T P a s e s ,
t h e i m a g e is f o r m e d , a n d a film r e c o r d i n g d e v i c e . lead nitrate.
A n u m b e r of a c c e s s o r i e s a r e u s u a l l y p r o v i d e d : a
s t i g m a t i z e r t o c o r r e c t a n y a s t i g m a t i s m in t h e METAL-COATING UNDER V A C U U M
object lens, a n object stage which h a s 4 degrees T h i s t e c h n i q u e w a s d e v e l o p e d p r i m a r i l y for
of f r e e d o m , e t c . A p u m p i n g s y s t e m m a i n t a i n s e l e c t r o n m i c r o s c o p y . Bessis a n d L o c q u i n h a v e
t h e h i g h v a c u u m i n t h e c o l u m n . A n a i r lock d e m o n s t r a t e d its a p p l i c a b i l i t y t o l i g h t m i c r o s -
p r o v i d e s for s p e c i m e n s t o b e i n t r o d u c e d w i t h o u t copy. M o s t scanning electron microscopes can
b r e a k i n g t h e v a c u u m i n s i d e t h e c o l u m n (Figure e x a m i n e only metal-coated specimens. For a
1.111). S o m e e l e c t r o n m i c r o s c o p e s a r e fitted w i t h long time these instruments were separate a n d
m a r g i n a l p h a s e c o n t r a s t , d e r i v e d from t h e H e i n e bulky items. N o w a d a y s they are integrated into
t e c h n i q u e in l i g h t m i c r o s c o p y , a d a p t e d b y L o c - the microscope or designed as a n attachable
q u i n (1956) for e l e c t r o n m i c r o s c o p y . s t a g e for t h e l i g h t m i c r o s c o p e , t h u s e n a b l i n g t h e
Several special techniques which h a v e been specimens to be observed d u r i n g the coating.
a m p l y described in t h e specialist literature a r e A p p l i c a t i o n of m e t a l c o a t i n g s o b l i q u e l y is
used to p r e p a r e the specimens. W e shall merely k n o w n as shadowing, because the resulting im-
cite, a s a n e x a m p l e , t h e u l t r a m i c r o t o m e s w h i c h a g e h a s a c e r t a i n n e g a t i v e s h a d o w effect from
a r e u s e d t o m a k e u l t r a f i n e s e c t i o n s of o b j e c t s o n e side. S p e c i m e n s w h i c h h a v e been coated in
e m b e d d e d in p l a s t i c r e s i n s , u s i n g g l a s s o r t h i s w a y c a n b e o b s e r v e d i n b r i g h t field illu-
diamond-cutting blades. m i n a t i o n , in p h a s e c o n t r a s t , in i n t e r f e r e n c e c o n -
T h e b e a m p a t h s in a light microscope a n d in t r a s t , b y t r a n s m i s s i o n o r b y reflection (Figure
a n e l e c t r o n m i c r o s c o p e c a n b e c o m p a r e d (Figure 1.115).
1.112), a n d t w o e x a m p l e s of h i g h p o w e r e l e c t r o n T h e s a m e e q u i p m e n t c a n be used to m a k e
m i c r o s c o p y a r e s h o w n in Figures 1.113 a n d 1.114. r e p l i c a s o r i m p r e s s i o n s b y d e p o s i t i o n of a fine
102 Instruments and techniques

Figure 1.113. A delicate specimen of kaolinite seen at a


magnification of 2,150,000 diameters, at 120 kV. The area
within the rectangular frame shows distribution of reticular
planes in three directions at 120, with a spacing of
0.45 nm (Philips)

Figure 1.114. Excellent compensation of energy lost in


traversing a 2 thick preparation at 120kV using a
velocity filter; castor plant root, with selective impregnation
(N. Poux, . Carasso and P. Favard, Lab. de Botanique,
Ecole Normale Suprieure de Paris) (Philips)

Figure 1.115. Path of atoms in the course


of shadowing; the resulting effect
(Policard, Bessis and Locquin)

Plastic material

F i g u r e 1.116. Diagrammatic section


of blood corpuscles implanted on a
plastic film shown while peeling the
^- film (Policard, Bessis and Locquin)

film, w h i c h is t r a n s p a r e n t to e l e c t r o n s ; t h e o b - v o g u e before t h e a d v e n t of t h e s c a n n i n g e l e c t r o n
j e c t is s u b s e q u e n t l y d i s s o l v e d a w a y . T h e s e t e c h - m i c r o s c o p e . I t is still a d v i s e d for r o u n d o b j e c t s ,
n i q u e s h a v e b e e n e x a m i n e d in s p e c i a l i z e d p a p - b e c a u s e it gives a t r u e t h r e e - d i m e n s i o n a l m o u l d
e r s w h i c h d e a l w i t h t h e t e c h n i q u e s of e l e c t r o n of t h e i m a g e in t h e f o r m of a shell w h e n t h e
microscopy. o b j e c t is d i s s o l v e d a w a y . T h i s p r o v i d e s a g o o d
r e p r e s e n t a t i o n of its v o l u m e a n d relief, w h e r e a s
CARBON COATING the scanning microscope only explores the sur-
T h e t e c h n i q u e of c o a t i n g thick i n s u l a t e d o b j e c t s face of t h e s p e c i m e n . F u r t h e r m o r e , after t h e
w i t h a t h i n l a y e r of c a r b o n , b y m e a n s of a n a r c s p e c i m e n h a s b e e n m o u l d e d , it c a n b e o b s e r v e d
s t r u c k b e t w e e n t w o e l e c t r o d e s , enjoyed g r e a t in t h e light m i c r o s c o p e a s well a s in t h e e l e c t r o n
Microanalysis by electron microscopy 103

m i c r o s c o p e , b e c a u s e c a r b o n a b s o r b s well e v e n h a v e b e e n developed, d e p e n d i n g on the objects


in t h i n films (Figure 1.109). to be examined.
T h e p r i n c i p l e of t h e m e t h o d is a s follows: t h e
TRANSFERS, I M P R E S S I O N S A N D F R A C T U R E s l i d e o n w h i c h t h e s e c t i o n is p l a c e d is c o v e r e d
PLANES w i t h a film of p l a s t i c m a t e r i a l , o n w h i c h all t h e
T h e s e a r e t h e t h r e e m o s t p o p u l a r t e c h n i q u e s in r a i s e d p o r t i o n s of t h e s p e c i m e n a r e i m p r i n t e d a s
e l e c t r o n m i c r o s c o p y . All t h r e e w e r e o r i g i n a l l y d e p r e s s i o n s . T h e film is t h e n e x a m i n e d in t h e
i n t r o d u c e d in light m i c r o s c o p y a n d c a n still b e e l e c t r o n m i c r o s c o p e . T h e m o u l d i n g s a r e ex-
e x t r e m e l y useful if o n e k n o w s h o w t o h a n d l e a m i n e d without a n y further preparation, or
them. b e t t e r still, after s h a d i n g . A s i m p l e r v a r i a n t of
F o r e x a m p l e , o n e m a y n e e d to e x a m i n e a t h e m e t h o d , w h i c h gives m o r e faithful i m a g e s , is
f u n g u s w h i c h is p a r a s i t i c o n t h e surface of a leaf. t o s h a d e t h e p r e p a r a t i o n before m o u l d i n g a n d
O f course, o n e c a n m a k e a transverse section of t h u s remove the gold deposit, the shadowing
t h e leaf t o e x a m i n e t h e s t r u c t u r e of t h e f u n g u s material, together with the plastic, b u t without
a n d h o w it is i m p l a n t e d . H o w e v e r , for r a p i d t h e cells, w h i c h a r e t o o thick; t h i s is k n o w n as
e x a m i n a t i o n it is p r e f e r a b l e t o t r a n s f e r t h e s t r u c - pre-shading moulding.
t u r e s of t h e s u r f a c e . E i t h e r o n e c a n s w a b t h e T h e preparations are no longer true replicas
s u r f a c e of t h e leaf w i t h d i l u t e c o l l o d i o n a n d t h e n b u t s h a d o w e d cells, of w h i c h o n l y t h e s h a d o w i n g
r e m o v e t h e s u r f a c e film w h e n it h a s d r i e d , o r o n e m a t e r i a l is o b s e r v e d . S h a d i n g is d o n e before a n y
can use a plastic transparent adhesive tape, f u r t h e r o p e r a t i o n ; t h i s is t h e m o s t faithful
w h i c h is a p p l i e d to t h e surface a n d t h e n s t r i p p e d m e t h o d p o s s i b l e for e x a m i n i n g t h e surface of
off to r e m o v e t h e p a r a s i t e s . T h e s a m e m e t h o d d r i e d cells. P r e p a r a t i o n s p r o d u c e d in t h i s w a y
c a n b e u s e d t o s t u d y c o a t i n g s s u c h a s t h o s e of yield i m a g e s i n t h e e l e c t r o n m i c r o s c o p e , w h i c h
t h e fungi, in w h i c h c a s e t h e t r a n s f e r d e t a c h e s t h e are accurately superimposable on those which
s u r f a c e l a y e r of cells to a l l o w e x a m i n a t i o n of a r e o b s e r v e d in t h e l i g h t m i c r o s c o p e , after s h a -
p a r t i c u l a r cells, w h i l e p r e s e r v i n g t h e i r r e l a t i v e dowing. Here we describe two techniques that
p o s i t i o n s in t h e t i s s u e s . c a n be applied to isolated cellspre-shadowed
I m p r e s s i o n s a r e a v a r i a t i o n of t h e t r a n s f e r m o u l d i n g a n d u s e of a n i n t e r m e d i a t e s e p a r a t i n g
m e t h o d . I n s t e a d of t r y i n g to r e m o v e t h e s u r f a c e layer.
cells, a n i m p r i n t of t h e surface s t r u c t u r e is t a k e n Pre-shadowed moulding. T h e cells o n t h e slide
b y s t r i p p i n g . I n t h i s c a s e , s w a b b i n g w i t h collo- are shaded by the usual methods, and then the
d i o n is t h e o n l y m e t h o d w h i c h c a n b e u s e d t o s h a d o w i n g m a t e r i a l o n l y is t r a n s f e r r e d b y m e a n s
t r a n s f e r t h e profile of t h e s u r f a c e from p l a t e t o of a p l a s t i c m e m b r a n e . C e l l s s m e a r s a r e s h a -
c o v e r g l a s s , following i n t e r m e d i a t e o b l i q u e d o w e d w i t h g o l d in t h e u s u a l w a y ; t h e y a r e t h e n
s h a d i n g t o b r i n g o u t t h e relief. i m m e r s e d in a n 0 . 5 % f o r m v a r s o l u t i o n in c h l o r o -
T h e s e t w o m e t h o d s yield i n f o r m a t i o n a b o u t f o r m , t h e n q u i c k l y r e m o v e d a n d d r i e d in t h e
t h e s u r f a c e of t h e s p e c i m e n s ; f r a c t u r e s c a n b e v e r t i c a l p o s i t i o n . A r e c t a n g u l a r g r i d is t h e n
u s e d to e x a m i n e u n d e r l y i n g l a y e r s . T h e s i m p l e s t i n s c r i b e d w i t h t h e p o i n t of a n e e d l e o n t h e slide,
m e t h o d is t o freeze t h e s p e c i m e n en bloc t o t o e n a b l e o n e to c u t t h e f o r m v a r m e m b r a n e i n t o
solidify it, a n d t h e n f r a c t u r e it b y i n s e r t i n g a p i e c e s t h e size of t h e o b j e c t c a r r i e r in t h e
w e d g e , a r a z o r b l a d e o r t h e b l a d e of a knife, e l e c t r o n m i c r o s c o p e . T h e s e pieces a r e d e t a c h e d
a l o n g t h e line of l e a s t m e c h a n i c a l r e s i s t a n c e t o b y i m m e r s i n g t h e slide in a c r y s t a l l i z i n g d i s h
separate structures located at varying depths w h i c h is filled w i t h w a t e r . T h e p i e c e s float,
w i t h i n t h e s p e c i m e n . T h e s e will b e s e e n o n t h e e n t r a i n i n g t h e g o l d of t h e s h a d o w i n g a n d t h e
face of t h e f r a c t u r e a s h o l l o w s o r e l e v a t i o n s . ( F o r cells. T h e cells a r e e l i m i n a t e d b y d i g e s t i o n in a
d e t a i l s , t h e r e a d e r is referred to s p e c i a l i s t l i t e r a - 2 % t r y p s i n s o l u t i o n in w a t e r , t o w h i c h a few
ture devoted to the electron microscope.) d r o p s of c a u s t i c s o d a s o l u t i o n h a v e b e e n
a d d e d . T h i s s o l u t i o n h a s t o b e filtered before
MOULDING u s e . D i g e s t i o n is u s u a l l y c o m p l e t e d w i t h i n a b o u t
I n f o r m a t i o n a b o u t t h e relief a n d t h e s u r f a c e of h a l f a n h o u r a t 3 7 C . T h e pieces a r e t h e n
cells c a n b e o b t a i n e d b y t a k i n g m o u l d s of t h e i r r e c o v e r e d , w a s h e d t w i c e in distilled w a t e r , a n d
s u r f a c e , w h i c h is fairly t h i n ( C l a u d e , 1949). m o u n t e d on the object carriers. O t h e r enzymes
Secondary s h a d i n g m a y subsequently be used to o r c h e m i c a l s c a n b e u s e d for t h e d i g e s t i o n ,
e n h a n c e relief. S e v e r a l m e t h o d s of m o u l d i n g a l t h o u g h p e p s i n yields t h e b e s t a n d m o s t r a p i d
104 Instruments and techniques

r e s u l t s . A s i n d i c a t e d a b o v e , cells h a v e to b e fixed Javelle. T h e y can be observed by transmission


w i t h a fixative w h i c h d o e s n o t interfere w i t h electron microscopy.
d i g e s t i o n . If o s m i c a c i d is u s e d , for e x a m p l e , t h e
cells s h o u l d n o t b e i m p r e g n a t e d , a s t h i s w o u l d
Ultra high-voltage electron microscopy
m a k e d i g e s t i o n v e r y difficult.
T h e m e m b r a n e r e s u l t i n g from this p r o c e s s is T h e instruments developed at Toulouse by G.
t h i c k e r t h a n t h a t u s u a l l y u s e d a s s u p p o r t ; t h i s is D u p o u y et al. in p a r t i c u l a r , u s e v e r y h i g h vol-
n e c e s s a r y b e c a u s e of t h e relief of t h e s p e c i m e n , t a g e s to p e n e t r a t e t h e s p e c i m e n s . I t is u n f o r t u n -
w h i c h is i m p r i n t e d in n e g a t i v e relief o n t h e ate that these i n s t r u m e n t s are not very c o m m o n ,
s u p p o r t (Figure 1.116). in v i e w of t h e r e s u l t s w h i c h t h e y h a v e p r o v i d e d
Use of an intermediate separating layer. If it is in b i o l o g y . U n d o u b t e d l y t h i s t y p e of i n s t r u m e n t
i m p o s s i b l e to d i g e s t t h e cell, a s e p a r a t i n g l a y e r in t h e e x p e r t h a n d s of a biologist w o u l d yield a n
c a n b e u s e d ( B r i c k a a n d T a b u i s , 1950): a b u n d a n t h a r v e s t of d i s c o v e r i e s .

(1) A u n i f o r m l a y e r of m e t a l , e.g. a l u m i n i u m , is
a p p l i e d to t h e s p e c i m e n b y v a c u u m s p u t t e r - Ultraviolet e x c i t e d electron microscopy
i n g , p e r p e n d i c u l a r l y to t h e s u r f a c e .
Gauzit has demonstrated that ultraviolet
(2) T h e s p e c i m e n is s h a d o w e d w i t h g o l d b y t h e
p h o t o n s c a n d e t a c h e l e c t r o n s from t h e t a r g e t
r o u t i n e m e t h o d for a p r e - s h a d o w e d r e p l i c a .
objects. A n interesting image can be m a d e with
(3) T h e w h o l e p r e p a r a t i o n is c o v e r e d w i t h a
these electrons. This instrument has been de-
m e m b r a n e of s u i t a b l e t h i c k n e s s b y i m m e r -
veloped by Balzers.
s i o n in a 0 . 5 % s o l u t i o n of f o r m v a r in c h l o r o -
form.
(4) T h i s m e m b r a n e is c u t i n t o s m a l l r e c t a n g u - Proton m i c r o s c o p y
l a r pieces w i t h a s h a r p p o i n t . T h e slide is
C. M a g n a n has converted the electron micro-
i m m e r s e d carefully in d i l u t e h y d r o f l u o r i c
scope into a proton microscope by substituting
a c i d o r h y d r o c h l o r i c a c i d to a t t a c k t h e a l u -
p r o t o n s for t h e e l e c t r o n s a n d b y i n v e r t i n g t h e
m i n i u m a n d to r e l e a s e t h e f o r m v a r film
p o l a r i t y of t h e e l e c t r o s t a t i c s y s t e m to focus t h e s e
w h i c h floats to t h e surface, s u p p o r t e d b y
protons. W h e n we consider that wavelengths
b u b b l e s of g a s w h i c h a r e e v o l v e d .
associated with protons are incomparably smal-
(5) T h e films a r e c o l l e c t e d , to b e m o u n t e d after
ler t h a n t h o s e a s s o c i a t e d w i t h e l e c t r o n s , t h e
i m m e r s i o n in distilled w a t e r . T h i s m e t h o d
t h e o r e t i c a l r e s o l v i n g p o w e r of s u c h a m i c r o s c o p e
r e l e a s e s t h e cells w i t h o u t d e s t r o y i n g t h e m
s h o u l d b e m u c h g r e a t e r t h a n t h a t of a n e l e c t r o n
a n d yields a n i m a g e of t h e cell a n d of its
m i c r o s c o p e . H o w e v e r , a n e s s e n t i a l factor h a s
i m p r i n t . A l u m i n i u m is t h e m e t a l u s u a l l y
h i t h e r t o l i m i t e d t h e p r a c t i c a l u t i l i z a t i o n of t h e
u s e d , a l t h o u g h it h a s a r a t h e r c o a r s e - proton microscope. Protons have a m u c h greater
g r a i n e d s t r u c t u r e . T h i s t e c h n i q u e is less mass than electrons, hence the destructive pow-
faithful t h a n t h a t of p r e - s h a d o w e d m o u l d s , e r of p r o t o n s o n t h e m a t e r i a l is c o r r e s p o n d i n g l y
b e c a u s e s h a d o w i n g is effected t h r o u g h a n g r e a t e r . F u r t h e r m o r e , b e c a u s e t h e p e n e t r a t i o n is
i n t e r m e d i a t e l a y e r w h i c h d e c r e a s e s t h e relief i n v e r s e l y p r o p o r t i o n a l to t h e m a s s of t h e p a r t i -
a n d m a s k s t h e fine d e t a i l s . cles, t h e r e a r e n o b i o l o g i c a l o b j e c t s y e t k n o w n
w h i c h a r e sufficiently t r a n s p a r e n t to p r o t o n s to
FREEZE-ETCHING
p r o v i d e i m a g e s of a n y i n t e r e s t . W e s h o u l d n o t
p r e j u d g e t h e f u t u r e , h o w e v e r , a n d this i n s t r u -
T h i s t e c h n i q u e r e p r e s e n t s a s o r t of c o r r o s i o n of
m e n t m a y p o s s i b l y b e a useful a d j u n c t to t h e
frozen s t r u c t u r e s after f r a c t u r e . B r o n c h a r t a n d
electron microscope, when we have improved
D e m o u l i n (1970) u s e t h e following t e c h n i q u e :
o u r m a s t e r y of t h e t e c h n i q u e s of t h e p r o t o n
fresh fungal s p o r e s a r e i m m e r s e d for a d a y in a
1 microscope.
0.1 m o l p h o s p h a t e buffer, p H 7 a t 4 C , to
w h i c h 2 5 % glycerol is a d d e d . T h e p r e p a r a t i o n is
c o n c e n t r a t e d b y c e n t r i f u g a t i o n . T h e block,
Ion microscopy
p l a c e d o n g o l d s u p p o r t s , is fixed in l i q u i d F r e o n
2 2 . T h e s a m p l e s a r e t r e a t e d b y t h e t e c h n i q u e of I f e l e c t r o n s w e r e r e p l a c e d b y i o n s , ion m i c r o -
M o o r et al. in B a l z a r ' s a p p a r a t u s . T h e r e p l i c a s scopes could be built. T h e i r principal a d v a n t a g e
a r e w a s h e d in s u l p h u r i c a c i d a n d t h e n in E a u d e w o u l d b e t h a t it w o u l d b e p o s s i b l e to e x a m i n e
Microanalysis by electron microscopy 105

s u r f a c e s w h i c h e m i t i o n s d u e to t h e i r b e i n g a t a h a v e t h e s a m e m a s s a n d a r e t h e r e f o r e difficult to
h i g h t e m p e r a t u r e . T h e r e a d e r is r e f e r r e d t o t h e s e p a r a t e , a n d also b e c a u s e t h e i o n i c b e a m d a m -
p a p e r b y G a u z i t for f u r t h e r i n f o r m a t i o n . ages the sample. Nevertheless, the instrument
h a s b e e n v e r y useful to localize p r e c i s e l y t h e
R A M A N SPECTROSCOPY a l k a l i e a r t h s in b i o l o g i c a l s t r u c t u r e s .
T h i s is b a s e d o n e x c i t a t i o n of c e r t a i n m o l e c u l e s
b y a s o u r c e of i n t e n s e light. If t h e m o l e c u l e s SECONDARY IONIC EMISSION
h a v e lost t h e i r fluorescence, o r it h a s b e c o m e T h e s p e c i m e n is b o m b a r d e d w i t h p r i m a r y i o n s
w e a k e r , a diffuse r a y c a n b e s e e n . O n e m u s t b e w h i c h d e t a c h c e r t a i n a t o m s o r g r o u p s of a t o m s ,
careful to c h o o s e a n e x c i t i n g w a v e l e n g t h w h i c h of w h i c h s o m e a r e i o n i z e d ; t h e s e a r e a n a l y s e d b y
d o e s n o t h a v e a c a t a l y t i c effect o n m o l e c u l e s . I n m a s s s p e c t r o m e t r y . T h e i n s t r u m e n t c a n collect
t h e diffusion s p e c t r u m , in a d d i t i o n to t h e c e n t r a l all t h i s e m i s s i o n b y a n i o n i c o p t i c a l s y s t e m ; it
r a y of t h e s a m e w a v e l e n g t h a s t h e i n c i d e n t l i g h t , c a n filter it a n d f o r m a n i m a g e of t h e d i s t r i b u -
t h e r e a r e n u m e r o u s feebler s y m m e t r i c a l w a v e s . t i o n of a p a r t i c u l a r e l e m e n t .
T h e difference b e t w e e n t h e w a v e l e n g t h s of ex- I o n i c m i c r o w a v e s a r e e m p l o y e d in a s y s t e m
c i t a t i o n a n d e m i s s i o n a r e c h a r a c t e r i s t i c of t h e v e r y s i m i l a r to t h e l a t t e r , e x c e p t t h a t t h e i m a g e
m o l e c u l e s . T h e i n s t r u m e n t c o n n e c t e d to a is f o r m e d b y s c a n n i n g a n ionic b e a m of a r g o n o r
m i c r o s c o p e p e r m i t s a r e s o l u t i o n of 1 . oxygen. W h e n using negative or positive ions,
i o n s of t h e o p p o s i t e c h a r g e a r e e m i t t e d . Q u a l i t a -
R A M A N LASER M I C R O W A V E S tive a n a l y s i s is difficult b e c a u s e s e c o n d a r y i o n s
T h e u s e of R a m a n m o l e c u l a r m i c r o w a v e s is can carry one or more elementary charges, a n d
b a s e d o n a n a d a p t a t i o n of t h e R a m a n effect a t t h e y m a y interfere w i t h e a c h o t h e r in s e v e r a l
t h e U n i v e r s i t y of Lille a n d e x p l o i t e d c o m m e r - different w a y s . Q u a n t i t a t i v e a n a l y s i s is m o r e
cially b y J o b i n - Y v o n . I n p r i n c i p l e , t h e a p p a r - difficult in b i o l o g i c a l s y s t e m s w h o s e surfaces a r e
a t u s is s i m i l a r to t h e e l e c t r o n i c m i c r o w a v e sys- n o t flat.
t e m of C a s t a i n g o r t h e i o n i c m i c r o w a v e s y s t e m As with electronic microwaves, the a p p a r a t u s
of S l o d z i a n . A b e a m of p h o t o n s from a l a s e r r e a c t s to p a r t i c u l a r c h a r g e s w h i c h m u s t b e
i l l u m i n a t e s a p u n c t a t e field of t h e s a m p l e . T h e d i s s i p a t e d b y m a k i n g t h e surface c o n d u c t i n g . A
light, diffused b y t h e R a m a n effect, is u s e d to m e t a l l i c grill is d e p o s i t e d o n it, a n d it is
identify a n d localize p o l y a t o m i c s t r u c t u r e s p r e - observed through the mesh.
s e n t in t h e s a m p l e . O n e c a n u s e it in o n e of t w o
ways; either record the R a m a n spectrum on a NUCLEAR MICROSOUND
2
p o i n t of t h e s a m p l e of a b o u t 1 , o r f o r m a n A p a r t i c l e a c c e l e r a t o r c a n b e u s e d to p r o d u c e a
i m a g e of t h e o b j e c t for a r a y c h a r a c t e r i s t i c of a b e a m of i o n s of s m a l l d i a m e t e r ( P i e r c e , 1975).
p a r t i c u l a r s u b s t a n c e . T h e m a i n l i m i t a t i o n of t h e A n i n t e n s e b e a m of i o n s 3 in d i a m e t e r c a n
m e t h o d is d u e to t h e low i n t e n s i t y of t h e R a m a n i n d u c e a n u c l e a r r e a c t i o n in n u m e r o u s e l e m e n t s
effect. T h e r e c o r d i n g of a s p e c t r u m t a k e s a l o n g of l o w a t o m i c n u m b e r , of w h i c h c a r b o n , n i t r o -
t i m e , a n d i m a g e s f o r m lines freely. B u t t h e s e g e n a n d o x y g e n a r e a c c e s s i b l e w i t h difficulty to
d i s a d v a n t a g e s a r e of little i m p o r t a n c e c o m p a r e d o t h e r m e t h o d s of a n a l y s i s . I t c a u s e s elastic
with the information obtained. Locquin a n d diffusion a n d e m i s s i o n of X - r a y s . T h e m e t h o d
B o y a r (1978) w e r e a b l e to c h a r a c t e r i z e t h e s h o w s t h e d i s t r i b u t i o n of i s o t o p e s of n i t r o g e n
c a l c i t e in t h e p r o t r u d i n g s p h e r o i d s of M y x o m y - a n d o x y g e n , for e x a m p l e .
c t e s , w h i c h w e r e of t h e o r d e r of 1 in d i a - T h e e n e r g y t r a n s f e r r e d to t h e m o l e c u l e s of t h e
meter. s p e c i m e n is sufficient to s t i m u l a t e specific c h e m -
ical r e a c t i o n s , w h i c h a r e n o t p o s s i b l e b y o t h e r
IONIC MICROWAVES m e a n s . T h i s m e t h o d h a s a p o w e r f u l p o t e n t i a l for
S l o d z i a n d e v i s e d t h e a p p a r a t u s . I t s p r i n c i p l e is a new cytochemistry.
to a n a l y s e s e c o n d a r y i o n s e m i t t e d b y t h e s a m p l e
d u e to t h e i m p a c t of a n i o n i c b e a m . T h e diffe- PYROLYSIS FOR MASS SPECTROGRAPHY
r e n t c o n s t i t u e n t s of t h e s a m p l e a r e identified b y T h e L a m m a l a s e r m i c r o s c o p e of L e i b d t a n d
t h e i r m a s s in a s p e c t r o g r a p h . O n e c a n e i t h e r M a t h e y ( 1 9 7 8 ; 1979) e n a b l e s o n e to volatilize a
form a n i m a g e of a p a r t i c u l a r c o n s t i t u e n t , o r m i c r o c r y s t a l in situ in t h e t h a l l u s of a l i c h e n , a n d
r e c o r d its m a s s b y s p e c t r o m e t r y a t a p a r t i c u l a r t o a n a l y s e it w i t h a m a s s s p e c t r o m e t e r l i n k e d to
p o i n t of t h e s p e c i m e n . T h e i n s t r u m e n t is l i m i t e d t h e m i c r o s c o p e . I t is t h e m e t h o d of c h o i c e to
b y t h e fact t h a t s e v e r a l different s e c o n d a r y i o n s e x t e n d c h e m o t a x o n o m y a t t h e c e l l u l a r level, a s
106 Instruments and techniques

o n e c a n c h a r a c t e r i z e s u c h p i g m e n t s as x a n t h - g r e a t e r t h a n t h a t of t h e h i g h e r o s m o t i c p r e s s u r e ,
enes, nucleic acids, polysaccharides, amino- h e n c e t h e w a t e r v a p o u r p a s s e s from t h e low
s u g a r s , p h o s p h o l i p i d s , lipids a n d a m i n e s ( W e i j - p r e s s u r e to t h e h i g h p r e s s u r e d r o p s o n w h i c h it
m a n , 1977). condenses, increasing their length at the expense
of t h e o t h e r s .
Capillary tubes 6 - 8 m m long with a lumen
1 m m in d i a m e t e r a r e p r e p a r e d . S e v e n d i s c r e t e
1.12 Microdeterminations of d r o p s , s e p a r a t e d b y s m a l l a i r b u b b l e s (Figure
physical values 1.117) a r e i n t r o d u c e d i n t o e a c h t u b e , 4 d r o p s of
r e f e r e n c e s o l u t i o n a n d 3 d r o p s of t h e s o l u t i o n
w h o s e o s m o t i c p r e s s u r e is to b e d e t e r m i n e d . T h e
Microdetermination of osmotic pressure
capillary tubes are sealed at both ends a n d
T h e c o n c e p t of o s m o t i c p r e s s u r e h a s n e v e r b e e n m o u n t e d o n a slide w i t h p l a s t i c i n e o r w i t h d u
e x p l a i n e d in a fully satisfactory m a n n e r , e v e n for N o y e r ' s s o l d e r . T h e a s s e m b l y is t h e n i m m e r s e d
physicists. Indeed, H . C . Eyster wants the term i n t o a P e t r i d i s h full of w a t e r . O n e r e a d s it
t o b e s t a n d a r d i z e d a n d h a s p r o p o s e d t h e follow- i m m e d i