CALIBRATION OF
MICROSCOPES FOR
MAGNIFICATION AND
RESOLUTION
By A. W. Agar, Agar Scientific Limited, 66a Cambridge Road, Stansted, Bssex, CM24 SDA, UK.
With the rapidly inereasing emphasis on quantitative results, itis very
important to know what the magnification of a microscope really is.
‘The various available calibrations are described, together with their
limitations, It is furthermore important to ensure that. a microscope is
performing to specification, or at least to an adequate level for the
experiment in hand. The appropriate tests for resolution and
instrument performance are reviewed,
Keywords: Mieroseope, Calibration, Magnification, Resolution
henever we use any Kind of microscope,
W be it a research light microscope, a
scanning electron microscope ora TEM,
‘ye noe to know the magnification. The manuf
turers hepllly provide this information in ap
proximate form, and for many applications this
is all ve noed to know. However, as sean as any
work involving the dimensions ofthe specimen
under examination is undertaken the instrument
needs mave exact calibration,
Calibrating the light microscope
Measurements are normally made by using
sgaticule within Uhe eyepiece, which les in the
Fecal plane ofthe eye lens Hovevey, this grabcule
isonly a comparative measure ast assumes the
magnification ofthe objective lens and also the
tbe length ofthe microscope which in some in
struments can be varied, Its mecessary therfore
to place a calibrated seale in the objet position,
ad to pect the magnified image onto the plene
the eyepiece gratiule, so thatthe latter may
be calibrated. Sage mietometers may be either
tramparent and covered with a cover gas ort:
Nective and without a cover glass (for incident
Tight instruments) {fig. 1). The ealibration is
only valid fr the particular objective and eye lens
beng wed and for the pertcular tube length at
that time (ne assumes thatthe objective lens is
fully served int its mount and Unt the eye lens
is properly seated in the eyepiece tube.
Once this alvation has been carried out for
given pair of lenses, it will remain valid pro
vided that there is nochange in any ofthe mech
anieal parameters.
Arecently develop test specimen is silieon
chip which hasbeen electron beam waitten with
‘square mesh ones of approximetely 10 mito:
retves, and with thicker lines every 500 micro
| |
Figure 1
meters was primarily designed forthe SBM but
isfound very useful als for incident light mito
mops, 28 the contrast i good (ig. 3).
Calibrating the SEM
"The electron lenses project a demagnifed image
of the elatronsoutee on to the specimen under
‘examination, na seanning raster normally eon
trolled by electromagnetic deflector coils and
diven in synchronism with the display tube
raster, The magnfiation is determined by the
‘Ste of the scanned raster on the display tube
Aivided by the size ofthe rater onthe specimen.
It is clear that the magnification therefore
depends on the excitation ofthe scanning cols,
‘modified by any remanent magnetim o acide,
tal eapacitance. tis quite possible thatthe two
scan cnils wll nat yield exactly the same magni:
eatin, so giving vise to orthogonal distortion.
‘The sean on the display tube is also subject to
distortion and nonlinearity. Even after the stem
iscalibeated,thore will bono guarentee that the
calibration wil not ehange with time.
‘The magnification is alo rather sensitively de
pendent on the working distance (objetve lens
to specimen) whichis best determined in terms
cf Ue objective lens curent. The calibration must
take thi fetor into eecount.
Since the magnification range of @ scanning
letra mievazope starts within the range ofthe
light nzroseope but goes upto very high values,