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Introduction to Fluorescence Microscopy

Section Overview:

Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence, which is formally divided into two categories, fluorescence and phosphorescence, depending upon the electronic configuration of the excited state and the emission pathway. Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime. The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited state lifetime.

Review Articles

  • Brief Overview of Fluorescence

    Derived from our introductory sections in the Physics of Light and Color, this section provides short explanations of the important associated phenomena as well as several tutorials and a references listing.

  • Basic Concepts in Fluorescence

    When coupled to the optical microscope, fluorescence enables investigators to study a phenomena in cellular biology. Foremost is the analysis of intracellular distribution of specific macromolecules in sub-cellular assemblies.

Interactive Java Tutorials

  • Jablonski Energy Diagram

    Explore how electrons in fluorophores are excited from the ground state into higher energy states, and the events that occur as these molecules relax by photon emission to then fall back into the ground-level energy state.

  • Solvent Effects on Fluorescence Emission

    Many environmental factors affect fluorescence emission such as interactions between the fluorophore and solvent molecules. Examine relaxation effects and spectral shifts that occur as a function of solvent polarity.

  • Photobleaching

    Photobleaching occurs when a fluorophore loses the ability to fluoresce due to photon-induced chemical damage. Explore variations in photobleaching rates in single, dual, and multiply labeled fluorescence specimens.

Selected References

Reference Listing

The field of fluorescence microscopy is experiencing a renaissance with the introduction of new techniques such as confocal, multiphoton, deconvolution, and total internal reflection, especially when coupled to advances in chromophore and fluorophore technology. The reference materials listed below were utilized in the construction of the fluorescence section of the Molecular Expressions Microscopy Primer.

Contributing Authors

Brian Herman and Victoria E. Centonze Frohlich - Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229.

Joseph R. Lakowicz - Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland and University of Maryland Biotechnology Institute (UMBI), 725 West Lombard Street, Baltimore, Maryland 21201.

Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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