Contents

FEATURES

Microspectrophotometry -potential applications in clinical oncology, 13

BY ZBIGNIEW L. OLKOWSKI

Particle size analysis using differential interference contrast verified by the SEM, 22
BY JOHN W. BLUHM AND MARTIN N. HALLER

Physics of the cell membrane: part five, mechanisms involved in cancer, 37

BY A. KEITH BREWER AND RICHARD A. PASSWATER

light source for microscopy, 49

BY SAM LEBER

Hot-stage microscopy for characterizing thermally sensitive polymers, 54

BY SCOTT I. MORROW

The stereomicroscope-instrumentation and techniques, 61

BY GENE E. SCHLUETER AND WALTER E. GUMPERTZ

Electron spectroscopy for chemical analysis of airborne particulates, 77

BY GENE R. GRIEGER

Scanning transmission electron microscopy, 83

BY IAN W. DRUMMOND

Glass-fiber filter paper-versatile laboratory tool, 97

BY

DEPARTMENTS

s.r, AVERSO

Editor's page, 6
Attending the Pittsburgh Conference on Analytical Chemistry and Applied
Spectroscopy, 107
BY WILLIAM N. WHAM, KENNETH S. HALABY, AND PAUL M. MILLS

Visiting Altex 1976, London, 115

BY WILLIAM N. WHAM, KENNETH S. HALABY, AND PAUL M. MILLS

Scientific economics, 120

Laboratory note: a multihead teaching microscope,i23
BY ARTHUR BELL AND HERBERT LETTAU

New products, 125

Available literature, 141
Advertising index, 144

COMING

High-pressure liquid chromatography in pharmaceutical analysis

Progress in the enzymatic assay of bile acids and sterols
Process gel filtration: part one, from lab to process scale
Rapid solvent selection for circular thin layer chromatography
Modular-programmed data acquisition/analysis in research
Nutritional value of sodium chloride, inorganic

An educational gas chromatograph using a thermal conductivity detector

COVER Photomicrograph of carcinoma of the colon involving the uterus, viewed with the
Balplan microscope. Courtesy of Bausch & Lomb, Scientific Optical Products
Division, Optics Center.

VOLUME 8, NUMBER 4
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Copyright 1976 by International Scientific Communications, Inc. All rights reserved.
Reproduction in whole or in part without written permission from International Scientific
Communications,lnc. is prohibited.
ALBYBL 8 (4) 1146 (1976)

mID
2

APRIL 1976

By Gene E. Schlueter and Walter E. Gumpertz

The stereomicroscope
INSTRUMENTATION AND TECHNIQUES

HE STEREOMICROSCOPE. also called dissecting

microscope, is widely used in many branches of

the physical and biomedical sciences, as well as in
industry. Less descriptive terms such as "wide-field
microscope" and "binocular microscope" are occasionally applied to the stereomicroscope, but most microscopists agree that these latter terms should be reserved
for compound microscopes. Compound microscopes
may be monocular or binocular, but they always
produce nonstereo images.
As the name implies, stereomicroscopes provide
spatial, three-dimensional images, much as do the human
eyes. This image is erect and nonreversed. The range of
magnification of the stereomicroscope is relatively low,
reaching a maximum of about 250X. Correspondingly,
the depth of field is larger and the working distances are
longer than those of compound microscopes of comparable magnification. Normally, the field diameters
observed are wider than those attainable with compound
microscopes.' These characteristics of the stereomicroscope are valuable in cases where the interpretation of
the structure of three-dimensional objects is required,
where manipulation of the specimen or working space is
necessary, or where a wide field of view must be
observed, at low magnification? This in-depth perception, coupled with a wide range of accessories utilized in
different optical and mechanical combinations, make
these instruments extremely useful for a multitude of
applications.I
Figura 1 The Greenough stereomicroscope system. a diagram
of the beam path.

Development
Very early in the history of the microscope, it was
reasoned that since nature had given us two eyes and
three-dimensional vision, binocular observation could be
used to advantage in the microscope (Ref. 4, p. 28).
The first known binocular microscope with two objectives and two eyepieces was designed by Cherubin
d'Orleans in 1677 ,5 a primitive instrument by today's
standards, with no erecting prisms, but a beginning.
In the 19th century, great strides were made in the
development and improvement of binocular tubes. The
Mr. Schlueter and Mr. Gumpertz are with the Department of
Microscopy, Wild Heerbrugg Instruments, Inc.

first known successful binocular microscope with two

eyepieces and a single objective, the binocular compound microscope, is attributed to Ridell, in the United
States in 1851 (Ref. 4, p. 29), with further contributions
made by Wenham, Siedentopf (Ref. 4, p. 30), Ives (Ref.
4, p. 33), and others.
The first stereomicroscope similar to what we know
today was designed by Greenough in 1897, and first
manufactured by Zeiss, and later by Leitz, Spencer,
Bausch and Lomb and others." These instruments were
great improvements in that erecting prisms were built
into the binocular tubes, allowing for reversion of the
image to a "right-side-up" position. The purpose of this
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APRIL 1976

article is to discuss the modern design and usage of the

stereomicroscope and its accessories in order to give the
reader a better understanding of how to select the proper
instrument to meet one's requirements.
Optical design

In practice, there are only two basic stereomicro.

scope designs, the Greenough system and the common
main objective systems (CMO). In the Greenough
system, the optics consist of paired objectives and
eyepieces, allowing the objectives to be made compact
and slender, with the lower portion tapered. In the
CMO system, in addition to the paired objectives and
oculars, a single common main objective is shared (see
Figures 1 and 2). Either design may be equipped with a
zoom-type continuously variable magnification
changer, or with a step-type magnification changer.
Both the Greenough and CMO systems have inherent
advantages and disadvantages which have been well
summarized by Gander 7 ,8 and condensed below.
Greenough-type stereomicroscopes. The advantages
of these microscopes are twofold: I) Since the objective pairs are more compact, with their lower ends
tapered and slender, as little of the field of view as
possible is covered and the specimen remains readily
accessible. 2) Chromatic aberrations can be corrected
with less effort, making the system generally less
expensive.
The disadvantages include: I) The intermediate
images are inclined to the specimen plane and tilted
relative to each other so that only the median bands
are sharply focused at the same time. Peripheral
portions of the field cannot be in optimum focus for
both eyes simultaneously. This can lead to fatigue
during prolonged observations. 2) In order to take
photographs of flat specimens, either the specimen
must be tilted, or one of the beam paths must be tilted
relative to the specimen.
Common main objective-type stereomicroscopes.
Advantages of this system include: I) The intermediate
image planes are parallel to the object plane and there
is no tilt between them. Consequently, both fields of
view can be imaged sharply and uniformly so that
prolonged observation is less tiring. 2) AcceSsories for
drawing, projection and photomicrography can easily
be adapted.
The disadvantages are that since the two imaging
beam paths traverse the main objective obliquely, the
chromatic aberrations are more difficult and expensive
to correct, and the main objectives are relatively large.
A large portion of the imaging beam passes through
peripheral areas of the main objective, while it is the
objective center that has best optical corrections.
There are no universally accepted criteria for measuring, comparing and evaluating which system is best,

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Figure 2 The common main objective stereomicroscoae

tem, a schematic diagram of the beam path.

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and consequently the individual microscopist must

make a judgment based upon viewing his own specimens and his particular requirements.

Magnification
In the stereomicroscope, imaging is accomplished in
two separate compound microscope systems, each
consisting of an eyepiece and an objective. Two
separate images from slightly different viewing angles
are picked up by the observer's eyes and fused in the
brain into a single three-dimensional image of the
specimen. The total magnification achieved is the
product of the objective and the eyepiece magnifications.
One of the basic differences in stereomicroscopes
available from the various manufacturers is the method
used in changing magnification. In the simplest instruments, the objectives are permanently mounted and
the magnification can be changed only by varying the
eyepiece power. Other systems feature interchangeable
objectives, so that magnifications can be changed by"
removing a detachable clamp-on or screw-on objective
and replacing it by another, or by changing to a higher
or lower power eyepiece. More advanced systems allow
changes in magnification with a sliding objective
housing or by rotating a turret which mounts paired
objective sets of different magnifications.
The most advanced stereornicroscopes currently in
use have either a "zoom" or a "rotating drum" system
for changing magnification. The ZOom systems involve
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the power stays unchanged. Two iris diaphragms are

needed because there are two images formed in a
stereomicroscope, one for the left eye, the other for
the right. The role of the double iris is significant, not
only for visual ovservation but also in photomicrography where the observer's eye is replaced by the
camera. Here only one of the two imaging paths is
picked up by the camera. Consequently only one of
the two iris diaphragms is functional with the camera.
The effect of increased depth and concomitantly
improved contrast is very striking on the photomicrographs.
As may be expected, the closing of the iris also
affects the overall light intensity. Consequently, the
exposure time will be considerably longer for the
picture taken with a stopped-down camera. The actual
setting of the double iris must be determined by trial and
error. As the double iris is stopped down gradually, the
image shows more contrast, and by further curtailment, the image "degrades" rapidly. Previously thin
boundary lines appear like double lines, points appear
like disks, etc., and delicate detail structures disappear.
The experienced microscopist uses the double iris to
strike a balance between maximum detail and maximum contrast, knowing full well that maximum
contrast and detail are opposites and that only a
balance between the two results in optimum visibility
of the image.

Stereomicroscopes serve for three-dimensional observation and manipulation of magnified small objects
where the perception of depth and contrast is usually
more important than in compound microscope observation. Since this increase in depth and contrast can
only be obtained at the expense of resolving power,
stereomicroscopes with an adjustable double iris diaphragm prove most valuable in situations where maximum detail of the object must frequently be traded to
improve depth and contrast.
Illumination

Proper illumination of the specimen is crucial for

effective microscopy. Reflected and transmitted light
illumination are both useful. Often they may be
combined. By changing the angle of incidence of the
illumination, or the angle of transillumination. various
internal structures and components may become alternately extinct and visible. Most stereomicroscope
illuminators use either line voltage or low voltage lamps
with a collector lens to concentrate the light in the
specimen plane. In a good low voltage lamp the
concentration of the lightspot should be adjustable by
an integral filament focusing mechanism. and its
intensity should be controlled by means of a dimmer
or a step transformer.
Light intensity has long been the limiting factor for
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STEREOMICROSCOPE continued

critical microscope observations and photomicrography. Transillumination bases are now available
which incorporate 12 v/lOO w quartz-halogen bulbs,
and they serve for both dark-field and bright-field
illumination. In bright-field, objects appear colored, or
in some cases as nearly transparent shadows silhouetted
against a white background. Conversely, in dark-field
illumination the background is dark and the objects
appear as if they were self-luminous bodies, often
reminiscent of stars against the night sky. Dark-field is
most useful in differentiating structures of unstained
specimens suspended in liquid media."

Supports for stereomicroscope and specimen

Selecting the proper supports for the microscope
and the specimen depends upon the nature of the
specimen, the method of illumination, and the space
required for manipulation or ancillary equipment. Most
manufacturers supply reflected-light stands, transmitted-light stands, and swinging-arm stands. Swingarrn
stands may be either table-top pedestal-type stands or
so-called table-clamp stands; the latter clamp to the
edge of the table.
Normally, specimens for transmitted light observation are placed directly on the transparent stage plate
of the transmitted-light stand, or on a clear glass slide.
Reflected-light specimens are usually placed on an
70

: APRIL 1976

opaque stage plate of contrasting color or an opaque

metallic or plastic slide. Glide stages are available that
allow movement of the specimen in all plane coordinate directions, and some of them feature 3600
rotatability. They are equally useful for transmitted
and reflected light.

Length measuring
Length measurements can be made by the eyepiece
reticle, a circular glass plate engraved with a ruling and
inserted into the measuring eyepiece. Calibration of the
eyepiece reticle is carried out by placing a stage
micrometer under the microscope and comparing its
scale to gauge the size of the reticle. The divisions on
the reticle are equidistant and their value changes with
changing objective powers. The measuring eyepiece is
characterized by its focusing eye lens. It enables
observers with different eyesights to focus on the
measuring reticle while the microscope image is also in
sharpest focus.

Drawing with the stereomicroscope

The drawing attachment or camera lucida attachment is an accessory valued primarily by biomedical
illustrators. The camera lucida is also called "drawing

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Caring for the stereomicroscope

Cleanliness is one of the most important factors in
obtaining consistently good results with the microscope. Whenever it is not in use, it should be covered
with a dust cover or stored in a locker or protective
hood. Optics should be stored so they will not be
subject to excessive moisture and dessicants should be
used to absorb moisture. Bulbs for the illuminators
should not be touched directly by the hand but
protected by tissue or a clean cloth when placed in the
socket. Needless to say, lens surfaces must be kept
spotless. Where stereomicroscopes are focused by
rack-and-pinion, the gear box should be kept free from
dust and dirt Since the rack-and-pinion mechanism
supports the weight of the stereomicroscope, this is
generally the first part of the stereomicroscope to wear
out since it is constantly under stress and tension.

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tube" or "tracing tube" because of the tubular

horizontal piece which protrudes to the right or to the
left of the instrument. With it, pencil tracing can be
done conveniently by both right-handed and lefthanded operators.
For a comprehensive discussion of camera lucida
attachments for binocular microscope viewing and
simultaneous tracing, see Refs. 10 and 11.

OUf

Proper tension adjustment can save costly repair bills.

hence never force the focusing movement
References
I. SCHLUETER, G.E.. "Manipulation of depth of field in

microscopy." Amer.l.ab. 7(4),65 (1975).

2. SCHLUETER, G.E. and GUMPERTZ. w" "Increase the
effectiveness of your stereomieroscopes." Evaluation Eng.,
17-21 (July/August 1971).
3. NEEDHAM, N.H .. The Practical Use of the Microscope (c.
Thomas Publishing Co .. Springfield. Ill.. 1958) p. 81 -2.
4. GAGE. S.H., The Microscope. 15th cd. (Comstock Publishing Co. Ithaca, 1932).
5. SCHLUETER, G.E .. "The origins and development of
microscopy." Amer. Lab.. 2. 53-61 (April 1970).
6. NEEDHAM. N.H . The Practical Use of the Microscope (e.
Thomas Publishing Co .. Springfield. III. 1958), p. 64.
7. GANDER, R., "Stereornicroscopes and three-dimensional
vision," Microskopion 24, Wild Hcerbrugg Ltd. Hecrbrugg,
Switzerland, p. 15-16 (1974).
8. GANDER, R . "Criteria of quality in microscope optics."
Microskopion 26. Wild Heerbrugg Ltd. Heerbrugg. Switzerland. p. 16,17 (1975).
9. SCHLUETER. G.E .. "A high intensity transmitted light
base for dark-field stereo microscopy ." A mer. Lab. 6 (4),40
(1974 ).
10. GRAY. P.. Encyclopedia of Microscopy and Microtechnique (Van Nostrand-Reinhold, Princeton, 1973) p. 4446.
11. GUMPERTZ, W., "Savings, time. and labor," Ind. Res.,
74 (May 1975).

AMERICAN LABORATORY

71