Conventional Light Microscopy

Eric S. Cole1

1 St. Olaf College, Northfield, Minnesota
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 9.1
DOI:  10.1002/9780470089941.et0901s12
Online Posting Date:  May, 2016
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In this molecular day and age, microscopy seems to be a neglected field of instruction. Too often professors, who themselves are strangers to the use of the light microscope, may hurry through a laboratory exercise designed to familiarize students with its uses. This chapter is designed to serve as a useful reference for instructors, perhaps demystifying some features of the microscope, and making their applications more user‐friendly and exciting. © 2016 by John Wiley & Sons, Inc.

Keywords: microscopy; lens; magnification; focus; resolution; refraction; phase‐contrast

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Table of Contents

  • Parts of the Light Microscope
  • Care and Maintenance
  • Basic Principles and Definitions
  • Magnification versus Resolution
  • Getting Comfortable
  • Finding the Object to be Viewed
  • Marking the location of an Object: Secrets of the Microscope Stage
  • A Quick Guide to Choosing from Various Optical Techniques
  • Köhler Illumination: Secrets of the Substage Condenser
  • Oil Immersion
  • Dark‐Field, Rheinberg, Polarized‐Light, Phase‐Contrast, and DIC Microscopy
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

   Abramowitz, M. 1985. Contrast Methods in Microscopy. Vol. 2. Olympus America Inc., Center Valley, Pa.
   Allen, R.D. , David, G.B. , and Nomarski, G. 1969. The Zeiss‐Nomarski differential interference equipment for transmitted‐light microscopy. Z. Wiss. Mikrosk. 69:193‐221.
   Cole, E.S. , Stuart, K.R. , Marsh, T.C. , Aufderheide, K. , and Ringlien, W. 2002. Confocal fluorescence microscopy for Tetrahymena thermophila . Meth. Cell Biol. 70:337‐359.
   Köhler, A. 1893. A new system of illumination for photomicrographic purposes. Z. Wiss. Mikroskopie 10:433‐440. Translated in Royal Microscopical Society‐Köehler Illumination Centenary, 1994.
   Murphy, D.B. 2001. Fundamentals of Light Microscopy and Electronic Imaging. Wiley‐Liss, New York.
   Newton, R.H. , Haffagee, J.P. , and Ho, M.W. 1998. Creating color contrast in light microscopy of living organisms. J. Biol. Education 32:29‐33.
   Nomarski, G. 1955. Microinterféromètre différentiel à ondes polarisées. J. Phys. Radium 16:9S‐11S.
   Omoto, C.K. and Folwell, J.A. 1999. Using darkfield microscopy to enhance contrast: An easy and inexpensive method. American Biol. Teacher 61:621‐624.
   Padawer, J. 1968. The Nomarski interference‐contrast microscope. An experimental basis for image interpretation. J. Royal Microscopical Society 88:305‐349.
   Salmon, E.D. , von Lackum, K. , and Canman, J.C. 2005. Proper alignment and adjustment of the light microscope. Curr. Protoc. Microbiol. 0:2A.1.1‐2A.1.31.
   Zernicke, F. 1955. How I discovered phase contrast. Science 121:345‐349.
Key References
   Barker, K. 2005. At the Bench: A Laboratory Navigator. Updated Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  A useful (brief) practical guide.
   Murphy , 2001. See above.
  A recent book (the best in my opinion, which does a good job discussing practical microscopy and its theoretical underpinnings.
Internet Resources
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