Proper Alignment and Adjustment of the Light Microscope

Edward D. Salmon1, Kate von Lackum2, Julie C. Canman1

1 University of North Carolina, Chapel Hill, North Carolina, 2 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2A.1
DOI:  10.1002/9780471729259.mc02a01s00
Online Posting Date:  October, 2005
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Abstract

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.

     
 
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Table of Contents

  • Major Components of the Light Microscope
  • Basic Imaging and Köhler Illumination Light Paths for Bright‐Field, Fluorescence, and Dark‐Field Microscopy
  • Basic Imaging for Dark‐Field Microscopy
  • Basic Protocol 1: Alignment for Köhler Illumination in Bright‐Field, Transmitted Light Microscopy
  • Basic Protocol 2: Alignment of the Eyepieces
  • Basic Protocol 3: Alignment for Köhler Illumination in Epifluorescence Microscopy
  • Basic Protocol 4: Alignment for Phase‐Contrast Microscopy
  • Basic Protocol 5: Alignment for DIC Microscopy
  • Alignment for Dark‐Field Microscopy
  • Basic Protocol 6: Alignment for Low‐Power Magnification Dark‐Field Microscopy
  • Basic Protocol 7: Alignment for High‐Power Magnification Dark‐Field Illumination
  • Support Protocol 1: Matching Microscope Magnification to Detector Resolution
  • Support Protocol 2: Calibrating Image Magnification with a Stage Micrometer
  • Tests for the Optical Performance of the Microscope
  • Support Protocol 3: Testing Phase‐Contrast and DIC Using Diatom Testing Slide
  • Support Protocol 4: Testing Phase‐Contrast, Dark‐Field, and DIC Microscopes Using a Squamous Cheek Cell Test Slide
  • Support Protocol 5: Testing Fluorescence Using a Red, Green, and Blue Fluorescent Tissue Culture Cell Test Slide
  • Support Protocol 6: Care and Cleaning of Microscope Optics
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

Literature Cited

Literature Cited
   Agard, D.A., Hiraoka, Y., Shaw, P., and Sedat, J.W. 1989. Fluorescence microscopy in three dimensions. Methods Cell Biol. 30:353‐377.
   Aldrich H.C. and Todd, W.J. 1986. Ultrastructure techniques for microorganisms. Plenum Press, New York.
   Allen, R.D., David, G., and Nomarski, G. 1969. The Zeiss‐Nomarski differential interference equipment for transmitted‐light microscopy. Z. Wiss. Mikf. Microtech. 69:193‐221.
   Carrington, W.A., Lynch, R.M., Moore, E.D.W., Isenberg, G., Forarty, K.E., and Fay, F.S. 1995. Superresolution three‐dimensional images of fluorescence in cells with minimal light exposure. Science 268:1483‐1487.
   Denk, W., Delatner, K.R., Gelperin, A., Keleinfeld, D., Strowbridge, B.W., Tank, D.W., and Yuste, R. 1994. Anatomical and functional imaging of neurons using 2‐photon laser scanning microscopy. J. Neurosci. Methods 54:151‐162.
   Faine, S. 1961. Simple teaching method for illustrating the principles of darkfield illumination. J. Bacteriol. 81:159‐160.
   Faine, S., Adler, B., Bolin, C., and Perolat, P. 1999. Leptospira and Leptospirosis, 2nd ed. MediSci, Melbourne, Australia.
   Hayden, J.E. 2002. Adventures on the dark side: An introduction to darkfield microscopy. Biotechniques 33:756‐761.
   Heim, R. and Tsien, R. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6:178‐182.
   Herman, B. 2002. Fluorescence Microscopy. In Current Protocols in Cell Biology (J. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott‐Schwartz, and K.M. Yamada, eds.) pp. 4.2.1‐4.2.10. John Wiley & Sons, Hoboken, N.J.
   Herman, B. and Jacobson, K. 1990. Optical Microscopy for Biology. Wiley‐Liss, New York.
   Hinsch, J. 1998. Mating camera to microscopes. Methods Cell Biol. 56:147‐152.
   Inoué, S. 1989. Imaging unresolved objects, super‐resolution and precision in distance measurement with video microscopy. Methods Cell Biol. 30:112.
   Inoué, S. and Oldenbourg, R. 1995. Chapter 17 In Handbook of Optics vol. 2 (Optical Society of America; M. Bass, ed.). McGraw‐Hill, New York.
   Inoué, S. and Spring, K. 1997. Video Microscopy. 2nd ed., Plenum Press, New York.
   Keller, H.E. 1995. In Handbook of Biological Confocal Microscopy (J.B. Pawley, ed.) 2nd ed., pp. 111‐126. Plenum, New York.
   Keller, H.E. 1997. Contrast enhancement in light microscopy. In Current Protocolsin Cytometry (J.P. Robinson, Z. Darzynkiewicz, P.N. Dean, A. Orfao, P.S. Rabinovitch, C.C. Stewart, H.J. Tanke, and L.L. Wheeless, eds.) pp. 2.1.1‐2.1.11. John Wiley & Sons, New York.
   Lawson, D. 1972. Photomicrography. Academic Press, London.
   Keller, H.E. 1998. Proper Alignment of the Microscope. Methods Cell Biol. 56:135‐146.
   Pawley, J. (ed). 1995. Handbook of Biological Confocal Microscopy, 2nd ed., Plenum, New York.
   Pluta, M. 1988. Advanced Light Microscopy, Vol. I: Principles and Basic Properties. Elsevier/North‐Holland, Amsterdam.
   Pluta, M. 1989. Advanced Light Microscopy Vol. II: Specialized Methods. Elsevier/North‐Holland Amsterdam.
   Rizzuto, R., Carrington, W., and Tuft, R. 1998. Digital imaging microscopy of living cells. Trends Cell Biol. 8:288‐292.
   Rost, F. and Oldfield, R. 2000. Photography with a Microscope. Cambridge University Press, Cambridge.
   Salmon, E.D. and Tran, P. 1998. High resolution video‐enhance differential interference contrast (VE‐DIC) light microscopy. Methods Cell Biol. 56:153‐183.
   Salmon, E.D., Shaw, S.L., Waters, J., Waterman‐Storer, C.M., Maddox, P.S., Yeh, E., and Bloom, K. 1998. A high‐resolution multimode digital microscope system. Methods Cell Biol. 56:185‐215.
   Sluder, G. and Wolf, D.E. (eds). 1998. Video microscopy. Methods Cell Biol. vol. 56.
   Smith, C. L. 1997. Basic confocal microscopy. In Current Protocols in Neuroscience (J.N. Crawley, C.R. Gerfen, R. McKay, M.A. Rogawski, D.R. Sibley, and P. Skolnick, eds.) pp. 2.2.1‐2.2.13. John Wiley & Sons, New York.
   Spencer, M. 1982. Fundamentals of Light Microscopy. Cambridge University Press, Cambridge.
   Sullivan, K.F. and Kay, S.A. (eds.) 1998. Green Fluorescent Proteins: Methods in Cell Biology, Vol. 58. Academic Press, San Diego.
   Svoboda, K. and Block, S.M. 1994. Biological applications of optical forces. Annu. Rev. Biophys. Biomol. Struct. 23:247‐285.
   Taylor, D.L. and Salmon, E.D. 1989. Basic fluorescence microscopy. Methods Cell Biol. 29:207‐237.
   Taylor, D.L. and Wang, Y.‐L. (eds). 1989. Fluorescence Microscopy of Living Cells in Culture. Methods Cell Biol. Vols. 29 and 30.
   Taylor, D.L., Nederlof, M., Lanni, F., and Waggoner, A.S. 1992. The new vision of light microscopy. Am. Sci. 80:322‐335.
   Zernike, F. 1942. Phase contrast: A new method for the microscopic observation of transparent objects. Physica 9:686‐693.
   Zernike, F. 1955. How I discovered phase contrast. Science 121:345‐349.
   Zernike, F. 1958. The wave theory of microscope image formation. In Concepts in Classical Optics (J. Strong, ed.) pp. 525‐536. W.H. Freeman, San Francisco.
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