Fluorescence Microscopy

Brian Herman1

1 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.2
DOI:  10.1002/0471143030.cb0402s13
Online Posting Date:  February, 2002
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Abstract

Absorption of energy as light by some molecules and emission of energy as fluorescence can occur only at certain wavelengths, which are characteristic for a given molecule (fluorophore). Fluorescence microscopes are equipped to observe the fluorescence of one or more specific fluorophores to localize specific molecules and analyzed cellular structures. This unit discusses the optics and other components of the fluorescence microscope, their functions and adjustments, and the digital darkroom.

     
 
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Table of Contents

  • Fluorescence Microscope Optics
  • Components of the Fluorescence Microscope
  • The Digital Darkroom
  • Suggested Reading
  • Figures
  • Tables
     
 
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Materials

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Figures

Literature Cited

Suggested Reading
   Abramowitz, M. 1993. Fluorescence Microscopy: The Essentials. Olympus‐America, New York.
   Adams, S.R. and Tsien, R.Y. 1993. Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 55:755‐784.
   Becker, E. 1989. Fluorescence Microscopy. Wild Leitz, Wetzlar, Germany.
   Heim, R. and Tsien, R.Y. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6:178‐182.
   Herman, B. 1998. Fluorescence Microscopy. BIOS Scientific Publishers, Oxford.
   Herman, B. 1989. Resonance energy transfer microscopy. Methods Cell Biol. 30:219‐243.
   Kapitza, H.G. 1996. Microscopy From the Very Beginning., Carl Zeiss Jena, Germany.
   Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy. Plenum, New York.
   Mason, W.T. 1993. Fluorescent and Luminescent Probes for Biological Activity. Academic Press, London.
   Nuccitelli, R. (ed.) 1994. A Practical Guide to the Study of Calcium in Living Cells. Methods Cell Biol. vol. 40.
   Pawley, J. 1995. Handbook of Biological Confocal Microscopy, 2nd ed. Plenum, New York.
   Periasamy, A. and Herman, B. 1994. Computerized microscopic vision in cell biology. J. Comput. Assisted Microsc. 6:1‐26.
   Pesce, A.J., Rosen, C.‐G., and Pasby, T.L. 1971. Fluorescence Spectroscopy: An Introduction for Biology and Medicine. Marcel Dekker, New York.
   Piston, D.W., Masters, B.R., and Webb, W.W. 1995. Three‐dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two‐photon excitation laser scanning microscopy. J. Microsc. 178:20‐27.
   Reichman, J. 1994. Glossary of Related Terminology with Page References to the Handbook of Optical Filters for Fluorescence Microscopy. Chroma Technology Corporation, Brattleboro, Vt.
   Reichman, J. 1994. Handbook of Optical Filters for Fluorescence Microscopy. Chroma Technology Corporation, Brattleboro, Vt.
  Slavik, J. (ed.) 1996. Fluorescence Microscopy and Fluorescent Probes. Plenum, New York.
   Taylor, D.L. and Wang, Y.L. (eds.) 1989. Fluorescence Microscopy of Living Cells in Culture, parts A and B. Methods Cell Biol. vols. 29 and 30.
   Tsien, R.Y. 1994. Fluorescence imaging creates a window on the cell. Chem. Eng. News 72:34‐36.
   Wu, P. and Brand, L. 1994. Resonance energy transfer: Methods and applications review. Anal. Biochem. 218:1‐13.
   Yguerabide, J. 1972. Nanosecond fluorescence spectroscopy of macromolecules. Methods Enzymol. 26:498‐578.
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