Principles and Application of Fluorescence Microscopy

Donald Coling1, Bechara Kachar2

1 University of California Berkeley, Berkeley, California, 2 National Institute on Deafness and Other Communication Disorders, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.10
DOI:  10.1002/0471142727.mb1410s44
Online Posting Date:  May, 2001
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Abstract

This overview discusses the principle of fluorescence along with practical discussions of fluorescent molecular probes, filters and filter sets, multiband filters and multi‐dye fluorescence, light sources, objective lenses, image resolution and the point‐spread function, fluorescence microscopy of living cells, and immunolabeling.

     
 
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Table of Contents

  • Fluorescent Molecular Probes
  • Filters and Filter Sets
  • Multiband Filters and Multidye Fluorescence
  • Light Sources
  • Microscope Objectives
  • Image Resolution and the Point‐Spread Function (PSF)
  • Fluorescence Microscopy of Living Cells
  • Immunolabeling: General Steps for Labeling Fixed Cells and Tissues
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

Videos

Literature Cited

   Carrington, W.A., Lynch, R.M., Moore, E.D.W., Isenberg, G., Fogarty, K.E., and Fay, F.S. 1995. Superresolution three‐dimensional images of fluorescence in cells with minimal light exposure. Science 268:1483‐1487.
   Dulbecco, R. and Vogt, M. 1954. Plaque formation and isolation of pure lines of poliomyelitis viruses. J. Exp. Med. 98:167‐182.
   Francon, M. 1961. Progress in Microscopy. Row, Peterson, Evanston, Ill.
   Haugland, R.P., Spence, M.T.Z., and Johnson, I.D. 1996. Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Eugene, Ore.
   Linscott's Directory of Immunological and Biological Reagents (updated quarterly). W.D. Linscott, Santa Rosa, Calif.
   Pawley, J.B. (ed.) 1995. Handbook of Biological Confocal Microscopy. Plenum, New York.
   Perkins, L.A., Hedgecock, E.M., Thomson, J.N., and Culotti, J.G. 1986. Mutant sensory cilia in the nematode Caenorhabditis elegans. Dev. Biol. 117:456‐487.
   Rost, F.W.D. 1992. Fluorescence Microscopy. Cambridge University Press, Cambridge.
   Spencer, M. 1982. Fundamentals of Light Microscopy. Cambridge University Press, Cambridge.
   Sternberger, L. 1979. Immunocytochemistry. John Wiley & Sons, New York.
Key Reference
   Inoue, S. and Spring, K.R. 1997. Video Microscopy. Plenum, New York.
  General reference for interested readers.
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