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Chapter 1 : Light Microscopy

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Light Microscopy, Page 1 of 2

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Abstract:

Light microscopy is made easy, interesting, and useful for bacteriological purposes if at least four different kinds of instruments are readily available, permanently set up for work, and maintained in good order. An additional instrument, a stereoscopic “dissecting” microscope, is valuable for undertaking the isolation of bacteria from nature on agar plates when colonies early in growth are small and close together. The chapter outlines the principles of basic microscope. Achieving high resolution as well as freedom from chromatic and spherical aberration requires attention to basic principles. Unfortunately, light of the most effective wavelength (namely, UV light of about 365 nm) is not perceived by the eye and does not pass through glass. The Koehler system of illumination is useful for high-resolution microscopy because it provides appropriate illumination of the field and makes good use of high-quality optics. The chapter outlines the steps allowing achievement of Koehler (nearly optimum) illumination for an oil-immersion objective. The chapter also focuses on dark-field microscopy, phase-contrast microscopy, interference microscopy, fluorescence microscopy, and photomicrography.

Citation: Murray R, Robinow C. 2007. Light Microscopy, p 5-18. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch1

Key Concept Ranking

Light Microscopy
0.59781176
Bright-field Microscopy
0.5881426
Confocal Microscopy
0.57478136
Fluorescence Microscopy
0.5473458
0.59781176
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Figures

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FIGURE 1

Basic light microscope and its parts. The assistance of R. Van Twest, Department of Microbiology, University of Guelph, is appeciated.

Citation: Murray R, Robinow C. 2007. Light Microscopy, p 5-18. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch1
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References

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1. Barer, R. 1968. Lecture Notes on the Use of the Microscope. Blackwell Scientific Publications, Oxford, United Kingdom. The advice of a master microscopist.
2. Boyde, A., 1990. Confocal optical microscopy, p. 185204. In P. J. Duke, and A. G. Michette (ed.), Modern Microscopies. Plenum Press, New York, NY. A description of equipment and applications.
3. Bradbury, S.,, and B. Bracegirdle. 1998. Introduction to Light Microscopy. Springer-Verlag, New York, NY..
4. Bradbury, S.,, and P. Everett. 1996. Contrast Techniques in Light Microscopy. Bios Scientific Publishers, Oxford, United Kingdom. Short handbook with simple explanations of lens systems.
5. Cargille, J. I. 1975. Immersion Oil and the Microscope. Technical reprint 101051. R. P. Cargille Laboratories, Cedar Grove, NJ. Another excellent booklet produced by a manufacturer.
6. Culling, C. F. A. 1974. Modern Microscopy—Elementary Theory and Practice. Butterworth & Co., London, United Kingdom. Another short paperback book.
7. Delly, J. G. 1998. Photography through the Microscope. Eastman Kodak Co., Rochester, NY. An example of the booklets produced by manufacturers involved in aspects of microscopy. These booklets are obtainable from such firms and their agents in updated versions and are generally excellent.
8. Engle, C. E. (ed.). 1968. Photography for the Scientist. Academic Press, Inc., New York, NY. General aspects of scientific photography, including photomicrography.
9. Hartley, W. G. 1993. The Light Microscope—Its Use and Development. Senecio Publishing Co., Oxford, United Kingdom.
10. James, J. 1976. Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff Medical Division, Amsterdam, The Netherlands. A fine book on theory and practice, with emphasis on the latter. It provides good advice on special and advanced techniques, including phase-contrast, interference, dark-field, polarization, and fluorescence microscopy.
11. McKinney, R. M.,, and W. B. Cherry,. 1985. Immunofluorescence microscopy, p. 891897. In E. H. Lennette,, A. Balows,, W. J. Hausler,, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 4th ed. American Society for Microbiology, Washington, D.C..
12. Mollring, F. K. 1981. Microscopy from the Very Beginning. Carl Zeiss, Oberkochen, Germany. Another excellent booklet produced by a manufacturer.
13. Pluta, M. 1988 to 1993. Advanced Light Microscopy, Vol. 1-3. Polish Scientific Publisher/Elsevier, Amsterdam, The Netherlands.
14. Quesnel, L. B., 1971. Microscopy and micrometry, p. 1103. In J. R. Norris, and D. W. Ribbons (ed.), Methods in Microbiology, vol. 5A. Academic Press Ltd., London, United Kingdom.
15. Quesnel, L. B., 1972. Photomicrography and macrophotography, p. 276358. In J. R. Norris, and D. W. Ribbons (ed.), Methods in Microbiology, vol. 7B. Academic Press Ltd., London, United Kingdom. Quesnel’s two articles are useful resources for optical details and practical advice on microscopy and photomicrography for microbiology in particular.
16. Richardson, J. H. 1991. Handbook for the Light Microscope. Noyes Publications, Park Ridge, NJ..
17.. Ross, K. F. A. 1967. Phase Contrast and Interference Microscopy for Cell Biologists. Edward Arnold, London, United Kingdom. An excellent explanation of the theory and practice of phasecontrast and interference microscopy. Also practical discussion of photographic techniques applied to microscopy.
18. Shillaber, C. P. 1944. Photomicrography in Theory and Practice. John Wiley & Sons, Inc., New York, NY. Nothing is likely to replace this classic text, which deals exhaustively but readably with the properties of objective lenses, oculars, and condensers. It sets out the practice of good illumination, weighs the advantages of different mounting media, and deals with both theoretical and practical bench microscopy; however, it antedates phase microscopy.
19. Shuman, H.,, J. M. Murray,, and C. Di Lullo. 1989. Confocal microscopy: an overview. BioTechniques 7:154613. This review includes an example of three-dimensional reconstruction.
20. Slayter, E. M. 1970. Optical Methods in Biology. John Wiley & Sons, Inc., New York, NY. A source book for the theoretical bases of most forms of microscopy and for analytical processes including diffraction, spectroscopy, and related optical techniques. It is concerned with principles and not practice.
21. Smith, R. F. 1990. Microscopy and Photomicrography—a Working Manual. CRC Press, Inc., Boca Raton, FL. A professionally illustrated procedural manual with minimal theory. Useful for a beginner with no experience.
22. Spencer, M. 1982. Fundamentals of Light Microscopy. Cambridge University Press, Cambridge, United Kingdom. A useful general survey.
23. Wang, Y.-L.,, and D. L. Taylor (ed.). 1989. Fluorescence Microscopy of Living Cells in Culture, part A. Fluorescent Analogs, Labelling Cells, and Basic Microscopy. Academic Press, Inc., New York, NY. A volume with helpful technical advice.

Tables

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TABLE 1

Common problems in light microscopy and their causes and remedies

Reprinted from reference with permission.

Citation: Murray R, Robinow C. 2007. Light Microscopy, p 5-18. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch1

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