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Contrast Enhancement in Light Microscopy
Optical microscopes, which are among the
oldest instruments of scientific discovery, continue to be key tools in both biomedical research and routine diagnosis. This remains true
despite the development of a wide range of new
imaging technologies, many with far greater
resolution—ranging from electron microscopes to the multitude of scanning probe systems available today. The overall simplicity of
optical microscopes, their minimally destructive impact, particularly on live specimens, and
the many microscopy techniques available for
enhancing or visualizing specific specimen features make this technique exceptionally useful
in biomedical applications. In recent years,
electronic imaging has both greatly enhanced
the capabilities of light microscopes and placed
ever-increasing demands on their optical performance.
A number of purely optical methods can be
used to enhance feature extraction from biological material for both visual observation and
subsequent electronic image processing. In the
present discussion, a short primer on image
formation in basic transmitted-light bright-field
microscopes and their resolution limits will be
followed by a brief outline of how to optimize
the imaging conditions by using what is known
as Köhler illumination, and finally by discussion of alternatives to bright-field microscopy.
KÖHLER ILLUMINATION
Köhler illumination is a method of focusing
and centering a microscope’s light source and
optics and setting its diaphragms to obtain the
best image detail and contrast. It was pioneered
in the late 1800s by August Köhler, a zoologist
in Giessen, Germany, who in 1900 joined Carl
Zeiss in Jena to head up the company’s microscopy group. Today, all manufacturers of highquality microscopes provide Köhler illumination in their instruments.
Basic Concepts
Understanding the possible contrast enhancement techniques and appreciating their
specific applications requires a clear grasp of
the basic concepts of Köhler illumination. It is
illustrative to follow the path taken by light
through a microscope from source to final image (see Fig. 2.1.1). A single, off-axis source
point is imaged by the collector into the front
focal plane of the condenser (condenser aperture diaphragm), where an image of the light
Contributed by H. Ernst Keller
Current Protocols in Cytometry (1997) 2.1.1-2.1.11
Copyright © 1997 by John Wiley & Sons, Inc.
UNIT 2.1
source is formed. Every point of this source
image is projected by the condenser as a parallel
pencil into infinity through the specimen. These
pencils uniformly distribute the light intensity
of each source point across their diameter,
which in turn is set by the field diaphragm.
In the back focal plane of the objective, also
called the objective exit pupil, the source is
imaged again and relayed by the eyepiece to its
exit pupil or eyepoint, which becomes the entrance pupil for the eye or imager. Four “sourceconjugated” planes in which a source image
appears can be identified, the latter two of which
are modulated by diffraction in the specimen.
Superimposed over this illumination beam
path is the image-forming beam path. The
specimen is conjugated to the field diaphragm
and imaged by the objective into the real intermediate image plane, then projected by the
eyepiece into infinity and reimaged by the detector’s objective (eye lens or camera lens) onto
the final sensor (retina, film, or camera).
There are several advantages to this dual
optical train. The field stop permits control over
internal stray light and internal reflections by
limiting the diameter of the illumination and
imaging bundles to that needed for a given
specimen field diameter. Contrast control can
be achieved by controlling the condenser aperture, which directly influences the coherence
and participation of diffracted wavefronts in the
formation of the final image: closing the condenser diaphragm increases contrast and decreases resolution. An experienced microscopist will attempt to find the best compromise
between resolution, contrast, and, to a lesser
extent, depth of field.
Most microscopes have built-in illuminators, so the steps for obtaining good Köhler
illumination are simple:
1. Achieving sharp specimen focus;
2. Achieving substage or condenser focus
and centering via the field diaphragm, and setting that diaphragm to cover a given objective’s
and eyepiece’s field of view; and
3. Setting the condenser aperture diaphragm for best contrast, resolution, and depth
of field, based on the nature of the specimen
itself.
Resolution and Contrast
The light path for Köhler illumination can
be explained by geometric optics, a way of
thinking about how light travels in a straight
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A
B
final image
exit pupil of
microscope
eye lens
eyepiece
real intermediate
image plane
exit pupil of
objective
objective
specimen
condenser
condenser
(aperture)
diaphragm
luminous field
diaphragm
collector
light source
Figure 2.1.1 Light path in Köhler illumination. (A) Path of the image-forming ray, with its four
conjugated planes. (B) Illumination path, again with four directly related or conjugated planes.
Contrast
Enhancement in
Light Microscopy
line as a ray. To understand the limits of resolution and the contrast transfer from object to
image, it is necessary to consider the electromagnetic wave nature of light, its wavelength
(λ), and the interaction of wavefronts with the
specimen’s structures. The theory of image formation through a microscope was developed by
Ernst Abbe, who distinguished between two
types of specimens: self-luminous objects
(light sources, luminescence, or fluorescence),
which follow the Rayleigh criterion, and illuminated objects, which follow the Abbe criterion. According to the Rayleigh criterion,
which is based on diffraction in the objective,
the point-to-point resolution, δ, (in µm) is given
by:
δ=
0.61λ
NA objective
In the case of an illuminated object, the Abbe
criterion defines the resolution, δ, as
δ=
λ
2NA
or
λ
NA objective + NA condenser
where λ is the wavelength of the emitted or
illuminating light and NA is the numerical
aperture of objective or condenser, given by the
2.1.2
Current Protocols in Cytometry
sine of half its collection angle times the refractive index of the medium between specimen
and objective.
With a self-luminous object, diffraction in
the objective itself causes the smallest image
point to expand into what is known as an Airy
disk (Fig. 2.1.2), whose diameter, D (in µml),
will be:
D=
1.22 λ
NA
With an illuminated object, diffraction is
caused by the structural features of the specimen and their spacing. The diffraction angle
(α) is determined by the wavelength and the
spacing or distance (d) between features.
sin α =
λ
d
The diffraction angle that an objective is
capable of collecting is directly related to its
numerical aperture. Abbe proved that for a
given structural spacing to be resolved, at least
two orders of diffracted light produced by this
spacing need to participate in the image forma-
tion. Interference between diffracted and nondiffracted wavefronts in the intermediate
image plane resolves structural detail and
determines the contrast at which the image is
rendered.
Bright-Field Microscopy
Bright-field microscopy is the optical technique most commonly used. With a microscope
set up and adjusted for Köhler illumination,
bright-field microscopy is ideally suited to the
study of specimens whose features are clearly
differentiated by differences in absorption.
Such specimens are also called “amplitude
specimens,” because they primarily change the
intensity or amplitude of the illuminating light.
Either inherent absorption or absorption induced by staining will change the gray level or
color contrast if spectral differences in absorption exist. The contrast appears against a white,
bright background.
Histochemically stained tissue sections of
all sorts, cytology and bacterial stains, and
naturally absorbing specimens are best studied
under a bright-field microscope.
A
Figure 2.1.2 (A) A typical Airy disk.
(B) Computer graph of the intensity
distribution of the Airy disk.
B
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optical terms, changes the mix of direct, nondiffracted wavefronts and diffracted light to
produce pseudorelief and enhanced contrast.
For more subtle enhancement, a similar effect
can be achieved by decentering the light source.
Care must be taken to retain even, uniform
illumination over the field.
Oblique illumination is a simple, inexpensive means to enhance contrast in unstained,
transparent sections, sediments, or casts and
can be a useful tool for finding focus in highly
transparent specimens.
OTHER MICROSCOPY
TECHNIQUES
Oblique and Anaxial Illumination
Just as the rising or setting sun will better
reveal the topography and mountain ridges of
a landscape than the noonday sun, obliquely
illuminating a specimen with limited internal
contrast can greatly enhance structural differences in optical density or refractive index and
turn an otherwise flat or almost invisible object
into an image of striking relief and apparent
three-dimensionality with clearly enhanced
contrast (Fig. 2.1.3).
To obtain oblique illumination with some
degree of reproducibility, a “turret condenser,”
which allows the condenser aperture diaphragm to be shifted laterally, is helpful. This
lateral displacement combined with the best
setting for the aperture’s diameter varies with
the objective’s numerical aperture and, in wave-
Dark-Field Illumination
Dark-field illumination greatly enhances a
microscope’s ability to detect minute structures
or particles, often far below the theoretical
limits of resolution—that is, even though the
size and spacing of the structures cannot be
resolved, their presence is obvious: they appear
bright on a dark (black) background. This dark
A
B
–1
0
+1
–1
–1
0
–1
0
+1
0
B´
+1
+1
G
B
Contrast
Enhancement in
Light Microscopy
Figure 2.1.3 Axial versus oblique illumination and the effects of aperture on resolution. (A)
Low-aperture axial illumination will not resolve structures that generate diffraction angles 0/+1 or
0/−1. (B) Shifting the same aperture to the side permits the objective to collect diffraction order 0
and −1, resolves the structure, and, with only one side band of diffracted light participating,
generates a relief effect.
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background is achieved by excluding all direct,
nondiffracted light from the objective. Specifically, the dark-field condenser produces a hollow cone of illumination with an aperture
higher than that of the objective. This can be
accomplished by an annular diaphragm in the
condenser aperture or by specific dark-field
condensers, such as “paraboloid” (dry) or
“cardioid” (oil) condensers (Fig 2.1.4). Objectives of high numerical aperture require a builtin iris or a funnel stop to reduce their numerical
aperture below that of the condenser. Because
the image is formed by diffracted light only, a
contrast reversal takes place. As a result, darkfield microscopy is exceptionally sensitive to
contamination; it is therefore imperative that
condenser, slide, and objective front lens be
perfectly clean. Contaminants, bacteria, cell
and urine casts, and blood, among others, lend
themselves well to dark-field studies.
Hoffman Modulation Contrast and
Varel Contrast
Hoffman modulation and Varel contrast
techniques are sophisticated methods for
oblique illumination. Images generated by
these methods exhibit a striking three-dimensional effect produced by converting directionally opposing specimen gradients in refractive
index or thickness into opposing gray-level
differences. The two techniques differ mainly
in the geometry of a special attenuator for the
diffracted light
direct light
specimen
internal mirror
condenser
annular stop
Figure 2.1.4 The dark-field condenser’s hollow cone of illumination passes by the objective. Only
light diffracted or refracted by the specimen is collected.
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nondiffracted, zero-order direct light in the
back focal plane of the objective and the corresponding illumination aperture in the condenser; the position of the attenuator determines the direction in which the gradients are
best contrasted.
In Hoffman modulation contrast a straight,
bar-shaped attenuator or modulator is placed
on the periphery of the objective’s aperture and
absorbs ~85% of the direct light coming from
a slit in the condenser aperture properly aligned
to superimposition over the modulator (Fig.
2.1.5).
In Varel contrast, an annular attenuator in the
very outer back aperture of the objective absorbs ~85% of the direct light coming from a
corresponding segment of an annulus in the
condenser. Only direct light is attenuated, while
diffracted light passes fully for a strikingly
improved contrast generation.
In both techniques, oblique brightfield can
be added when specimens of relatively high
inherent contrast are studied. Unstained live
tissue and cell cultures in either glass or plastic
vessels make ideal specimens for these techniques; the improved depth perception of the
resulting images also facilitates micromanipulation or microinjection.
Differential Interference Contrast
Differential interference contrast is the most
sophisticated, most flexible, and potentially
most highly resolving technique available; it
converts specimen gradients into gray-level differences and produces a striking pseudo-threedimensional effect. The system employs polarized light and special prisms called Wollaston
prisms to produce two slightly sheared or separated wavefronts, which traverse the specimen
(Fig. 2.1.6). The amount of shear is usually
below the resolution of a given objective and is
a function of the Wollaston prisms in the condenser and objective. Specimen gradients in
refractive index or thickness result in an optical
path difference between the two sheared wavefronts. When the wavefronts are recombined
and made to oscillate in a common plane by an
analyzer, different amounts of constructive or
destructive interference produce distinct graylevel differences for opposing gradients, with
the greatest contrast along the direction of
shear. The system can be set to maximum contrast for any specific specimen gradient by
adjusting one of the prisms or using a special
compensator. It is often desirable to use the full
objective and condenser apertures, particularly
when using video-enhanced imaging to extract
the very smallest contrast differences so as to
detect and visualize intracellular organelles
such as microtubules.
Differential interference contrast (largely
the version proposed by Nomarski) has contributed greatly to the study of live cells and tissues
and is now an indispensable tool in develop-
3% transmittance
100% transmittance
modular
15% transmittance
objective
specimen with
gradients
condenser
slit aperture
Contrast
Enhancement in
Light Microscopy
Figure 2.1.5 Basic principle of modulation contrast.
2.1.6
Current Protocols in Cytometry
mental biology, physiology, neuroscience, and
many other disciplines. Because it employs
polarized light, plastic specimen vessels should
not be used for this method, as they tend to show
birefringence and depolarize the sheared wavefronts. To avoid strain or stress in the condenser
and objective, it is important to use components
recommended by the manufacturer.
Phase Contrast
Phase contrast microscopy is designed for
the study of thin, unstained sections or live
cultures—i.e., transparent specimens with
minimal inherent contrast. Unlike amplitude or
absorbing specimens, for which the diffracted
wavefronts are phase-shifted by one-half of a
wavelength, such so-called phase specimens
generate shifts of only one-quarter of a wavelength. The interference conditions between
diffracted and direct wavefronts are neither
constructive nor destructive, and the image
contrast is poor. The Dutch physicist Frits
Zernicke won the Nobel prize for his proposal
to add to condenser and objective elements that
intermediate image
analyzer (135 )
second Wollaston prism
objective
specimen
condenser
first Wollaston prism
polarizer (45 )
Figure 2.1.6 Principles of differential interference contrast. The separation between the two
sheared beams is greatly exaggerated.
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shift the phase of nondiffracted, direct light by
one-quarter of a wavelength, and at the same
time attenuate the intensity so as to greatly
enhance the interference conditions for the image rendition. The result is an image wherein
“positive-phase” contrast areas of higher refractive index appear darker. Specific gray levels optically “stain” areas of specific refractive
index and thickness. This is accomplished by
an illumination annulus in the aperture plane of
the condenser along with a conjugate phase
ring in the back focal plane of the objective
that acts as both attenuator and phase shifter.
A green filter further enhances the contrast
(Fig. 2.1.7).
In contrast to differential interference or
oblique illumination techniques, which optically “stain” specimen gradients and generate
P´
λ/2
λ/4
P
R
Contrast
Enhancement in
Light Microscopy
Figure 2.1.7 Phase contrast. The annulus, R, in the condenser aperture is superimposed on the
phase plate behind the objective, which is both an attenuator and a phase shifter.
2.1.8
Current Protocols in Cytometry
a pseudo-three-dimensional effect, phase contrast produces a two-dimensional image of index- and thickness-specific gray levels.
The limitations of phase contrast are determined by the illumination aperture, a function
of the condenser’s phase ring, and by the “halo”
effect along steep specimen gradients, which
limits the section thickness for phase contrast
to ∼5 µm.
Reflection Interference
Reflection interference microscopy looks at
the interference pattern that is naturally present
between a cell or tissue and its substrate (cover
glass). Wavefronts reflected at the cell surface
interfere with those reflected at the substrate
and are one-half wavelength out of phase for
those areas where the cell adheres to the cover
glass, resulting in destructive interference and
darkness (adhesion plaques).
Using reasonably monochromatic incident
light obtained by filtering light from a tungsten
halogen or, better, a mercury lamp through a
green filter, along with good Köhler illumination, produces a striking contrast that allows
direct analysis of a cell’s proximity to the substrate on which it grows. Limiting the illumi-
nation aperture further contrasts intracellular
features based on the varying path differences
they generate. Video enhancement of the contrast can also considerably improve the results
(see Key References for suggestions for further
reading on this topic).
Polarized Light Microscopy
For a wide range of biological materials,
visualization can be considerably improved by
using either simple polarized light illumination
or polarization contrast, whereby a polarizer
below the condenser (usually oriented “eastwest”) linearly polarizes incoming wavefronts.
An analyzer behind the objective is oriented at
90o to the polarizer; without a specimen the
field of view is dark. Specimens with distinct
structural orientation, such as muscle, nerve, or
bone tissue, are birefringent—i.e., they display
different refractive indices in different directions. Depending on their thickness and orientation to the polarizer and analyzer, such specimens will alter the plane of vibration of incoming polarized light and change it to some form
of elliptically polarized light, part of which will
then be able to pass the analyzer. This results
in bright specimen images on a dark back-
real image plane
barrier filter
source
dichromatic
filter
exciting radiation
exciter
filter
objective
emitted radiation
object
Figure 2.1.8 Epifluorescence is made possible by a dichromatic filter that reflects the exciting
radiation down on the specimen and allows the emitted radiation to pass upward to the eye.
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ground. When the full visible spectrum of light
is used, specific wavelengths will be suppressed and others enhanced as a function of
the path difference the specimen has generated
between its two orthogonal vibration directions. This can result in vibrant interference
colors, which in turn provide information about
the specimen’s birefringence and thickness.
A detailed discussion of polarized light microscopy—especially the quantitative aspects
of the analysis of specimen birefringence and
directional and structural orientation—would
require far more in-depth treatment than can be
contained in this brief overview; the reader is
encouraged to consult the relevant literature
(see Key References) for further information.
Fluorescence Microscopy
One of the fastest-growing tools in biomedical microscopy is fluorescence. The exceptional sensitivity of this technique, combined
with the ever-growing list of very specific protein markers and fluorophores covering a wide
range of different colors that are available, have
made it indispensable for qualitative and quantitative diagnosis. Autofluorescent or fluorophore-labeled specimens are excited with
short-wavelength radiation and almost instantly convert some of the absorbed exciting
radiation to emitted longer-wavelength fluorescence.
In present-day microscopes, excitation is
provided almost exclusively by either incident
light or epiillumination. The light source is
usually a mercury or xenon gas discharge lamp.
Special filter/reflector combinations isolate the
fluorophore’s specific emission wavelength
from the exciting radiation to maximize the
efficiency of both and thereby produce a bright,
sharp fluorescent signal on a black background
(Fig. 2.1.8). Ideally the microscope should be
equipped with objectives of high numerical
aperture, magnifications just high enough to see
the areas of interest, and good chromatic correction. Much of the image capture is done with
sensitive cameras either in real time or, for low
light levels, with long-term signal integration.
A multitude of publications, textbooks, and
reprints detailing fluorescence microscopy are
available in bookstores and from most of the
major microscope manufacturers. For a more
in-depth look into these interesting and useful
microscope methods the reader is encouraged
to contact these sources (see Key References).
Contrast
Enhancement in
Light Microscopy
KEY REFERENCES
General Microscopy
Born, M. and Wolf, E. 1970. Principles of Optics.
Pergamon Press, Elmsford, N.Y.
Bradbury, S., Evennett, P.J., Haselmann, H., and
Piller, H. 1989. Dictionary of Light Microscopy.
Oxford University Press, Oxford.
Herman, B. and Jacobsen, K. 1990. Optical Microscopy for Biology. Wiley-Liss, New York.
Lacey, A.J., 1989. Light Microsopy in Biology: A
Practical Approach. IRL Press, Oxford.
Pawley, J. 1989. Handbook of Biological Confocal
Microscopy. Plenum, New York.
Pluta, M. 1988. Advanced Light Microscopy, Vol. I.
Elsevier Science Publishing, New York.
Pluta, M. 1989. Advanced Light Microscopy, Vol. II.
Elsevier Science Publishing, New York.
Pluta, M. 1992. Advanced Light Microscopy, Vol.
III. Elsevier Science Publishing, New York.
Spencer, M. 1982. Fundamentals of Light Microscopy. Cambridge University Press, Cambridge.
Oblique Illumination Techniques
Ellis, G.W. 1981. Edge Enhancement of Phase Phenomena. U.S. Patent No. 4255014.
Hoffman, R. 1975. The modulation contrast microscope. Nature 254:586-588.
Kachar, B. 1985. Asymmetric illumination contrast.
Science 22:766-768.
Hoffman Modulation Contrast
Hoffman, R. and Gross, L. 1975. Modulation contrast microscopy. Appl. Opt. 14:1169-1176.
Differential Interference Contrast
Allen, R.D., David, G.B., and Nomarski, G. 1969.
The Zeiss-Nomarski differential interference
equipment for transmitted light microscopy. Z.
Wiss. Mikrosk. Mikrosk. Tech. 69:193-221.
Francon, M. 1962. Progress in Microscopy. Row,
Peterson, Evanston, Ill.
Lang, W. 1979. Nomarski Differential Interference
Contrast Microscopy. Carl Zeiss, Oberkochen,
Germany.
Padawer, J. 1968. The Nomarski interference contrast microscope. J. Roy. Miscrosc. Soc. 88: 305349.
Phase-Contrast Microscopy
Francon, M. 1962. Progress in Microscopy. Row,
Peterson, Evanston, Ill.
Ross, K.F.A. 1967. Phase Contrast and Interference
Microscopy for Cell Biologists. Edward Arnold,
London.
Zernicke, F. 1942. Phase contrast, a new method for
the microscopic observation of transparent objects. Physics 9:686-693.
2.1.10
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Reflection Interference Microscopy
Beck, K. and Bereiter-Hahn, J. 1981. Evaluation of
reflection interference contrast images of living
cells. Microsc. Acta 84:153-178.
Gingell, D. and Todd, J. 1979. Interference reflection microscopy: A quantitative theory for image
interpretation. Biophys. J. 26:507-526.
Izzard, C.S. and Lochner, L.R. 1976. Cell to substrate contacts in living fibroblasts. J. Cell Sci.
21:129-159.
Ploem, J.S. 1975. Reflection Contrast Microscopy
as a Tool for Investigation of the Attachment of
Living Cells to a Glass Surface. Blackwell Scientific, Oxford.
Polarized Light Microscopy
Patzelt, W.J. 1985. Polarized Light Microscopy:
Principles, Instruments, Applications. E. Leitz,
Wetzlar, Germany.
Shurcliffe, W.A. 1962. Polarized Light. Harvard
University Press, Cambridge, Mass.
Waggoner, A.S., DeBiasio, R., Bright, G.R., Ernst,
L.A., Conrad, P., Galbraith, W., and Taylor, D.L.
1989. Multiple spectral parameter microscopy.
Methods Cell Biol. 30:449-478.
Photomicrography
Delly, J.G. 1980. Photography through the Microscope. Kodak Publication P-2. Kodak, Rochester, N.Y.
Loveland, R.P. 1981. Photomicrography: A Comprehensive Treatise. John Wiley & Sons, New
York.
Video Microscopy
Allen, R.D., Allen, N.S., and Travis, J.L. 1981.
Video enhanced contrast, differential interference contrast microscopy. J. Cell Mot. 1:298302.
Allen, R.D. and Allen, N.S. 1983. Video enhanced
microscopy with a computer frame memory. J.
Microsc. 128:3-7.
Shurcliffe, W. 1975. Polarized Light, Benchmark
Papers in Optics. John Wiley & Sons, New York.
Inoué, S. 1981. Video image processing greatly
enhances contrast quality and speed in polarization microscopy. J. Cell Biol. 89:346-356.
Fluorescence Microcopy
Inoué, S. 1986. Video Microscopy. Plenum, New
York.
Bright, G.R. 1993. Multiparameter imaging on cellular function interference. In Fluorescence
Probes for Biology Function of Living Cells: A
Practical Guide (W.T. Mason and G. Rolf, eds.)
pp. 204-215. Academic Press, San Diego.
Herman, B. and Lemasters, J.J. 1993. Optical Microscopy, Emerging Methods and Applications.
Academic Press, San Diego.
Taylor, D.L. and Wang, Y.L. 1989a. Fluorescence
Microscopy of Living Cells in Culture (Part A,
Vol. 29). Academic Press, San Diego.
Taylor, D.L. and Wang, Y.L. 1989b. Fluorescence
Microscopy of Living Cells in Culture (Part B,
Vol. 30). Academic Press, San Diego.
Inoué, S. 1987. Video microscopy of living cells and
dynamic molecular assemblies. Appl. Optics
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Inoué, S. 1988. Progress in video microscopy. Cell
Motil. Cytoskeleton 10:13-17.
Schotten, D. 1993. Electronic Light Microscopy.
Wiley-Liss, New York.
Weiss, D.G., Maile, W., and Wick, R. 1992. Video
microscopy. In Light Microscopy in Biology
(S.J. Lacey, ed.) pp. 221-278. IRL Press, Oxford.
Contributed by H. Ernst Keller
Carl Zeiss, Inc.
Thornwood, New York
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