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-

optical terms, changes the mix of direct, non-diffracted 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.

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

Axial Illumination Photography

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.

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.

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 built-in 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, dark-field 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 direction-ally 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 internal mirror condenser annular stop internal mirror condenser annular stop

Darkfield Condenser

diffracted light direct light-^

specimen

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.

Image Cytometry Instrumentation 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-three-dimensional 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 wave-fronts. 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 gray-level 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

Contrast Enhancement in Light Microscopy

Cardioid Microscope Condenser
Figure 2.1.5 Basic principle of modulation contrast.

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 wave-fronts. 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

Interference Contrast
Figure 2.1.6 Principles of differential interference contrast. The separation between the two sheared beams is greatly exaggerated.

Image Cytometry Instrumentation 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

Contrast Enhancement in Light Microscopy

Halo Effect Phase Contrast 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.

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 |m.

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 "east-west") 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 dichromatic filter objective object real image plane dichromatic filter objective object

Other Key Microscopy Techniques
emitted radiation

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.

Image Cytometry Instrumentation 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 fluoro-phore-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.

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.

Shurcliffe, W. 1975. Polarized Light, Benchmark Papers in Optics. John Wiley & Sons, New York.

Fluorescence Microcopy

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.

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.

Inoue, S. 1981. Video image processing greatly enhances contrast quality and speed in polarization microscopy. J. Cell Biol. 89:346-356.

Inoue, S. 1986. Video Microscopy. Plenum, New York.

Inoue, S. 1987. Video microscopy of living cells and dynamic molecular assemblies. Appl. Optics 26:3219-3225.

Inoue, 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

Image Cytometry Instrumentation

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