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Fluorescence Microscopy

Section Overview:

Fluorescence illumination and observation is the most rapidly expanding microscopy technique employed today, both in the medical and biological sciences, a fact which has spurred the development of more sophisticated microscopes and numerous fluorescence accessories. Epi-fluorescence, or incident light fluorescence, has now become the method of choice in many applications and comprises a large part of this tutorial. We have divided the fluorescence section of the primer into several categories to make it easier to organize and download. Please follow the links below to navigate to points of interest.

Review Articles

  • Introduction to Fluorescence Microscopy

    Learn the basic concepts of fluorescence, a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical, mechanical, or chemical mechanism.

  • The Fluorescence Microscope

    Unlike other modes of optical microscopy based on macroscopic specimen features, such as birefringence, fluorescence microscopy is capable of imaging the distribution of a single molecular species based solely on the properties of fluorescence emission.

  • Light Sources

    In order to generate enough excitation light intensity to furnish secondary fluorescence emission capable of detection, powerful light sources are needed such as mercury or xenon arc (burner) lamps.

  • Optimization and Troubleshooting

    Reviewed in this article are key features of fluorescence microscopy such as detecting fluorescent objects that can be faintly visible or very bright relative to the background, as well as common problems with microscope configuration.

  • Electronic Imaging Detectors

    The featured discussion is intended to aid in understanding the basics of light detection and to provide a guide for selecting a suitable detector for specific applications in fluorescence microscopy.

  • Introduction to Fluorophores

    Widefield fluorescence and laser scanning confocal microscopy rely on secondary fluorescence emission as an imaging mode, primarily due to the high degree of sensitivity afforded by the techniques.

  • Optical Highlighter Fluorescent Proteins

    Optical highlighters generally display little or no initial fluorescence under excitation at the imaging wavelength, but increase their fluorescence intensity after activation by irradiation at a different wavelength.

  • Fluorescence Photomicrography

    Read more about photomicrography under fluorescence illumination conditions can present a unique set of circumstances that may pose special problems for the microscopist.

  • Practical Aspects of Fluorescence Filter Combinations

    A wide spectrum of filter cubes is available from most major manufacturers, which now produce filter sets capable of imaging most of the common fluorophores in use today.

  • Glossary of Terms in Fluorescence and Confocal Microscopy

    The featured resource is provided as a guide and reference tool for visitors who are exploring the large spectrum of specialized topics in fluorescence and laser scanning confocal microscopy.

Advanced Techniques in Fluorescence Microscopy

  • Introduction to Confocal Microscopy

    Confocal microscopy offers the ability to control depth of field, elimination or reduction of background information away from the focal plane, and the capability to collect serial optical sections from thick specimens.

  • Multiphoton Excitation Microscopy

    Multiphoton fluorescence microscopy is a powerful tool combining the techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution and 3-D images of specimens.

  • Fluorescence Resonance Energy Transfer (FRET)

    When the technique of fluorescence resonance energy transfer (FRET) is applied to optical microscopy, it permits determination of the approach between two molecules within several nanometers.

  • Total Internal Reflection Fluorescence Microscopy

    TIRFM is commonly employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology.

  • Laser Systems for Optical Microscopy

    The lasers employed in optical microscopy are high-intensity monochromatic light sources, which are useful for many techniques including optical trapping, lifetime imaging studies, and photobleaching recovery.

  • Fluorescence & DIC Combination Microscopy

    Fluorescence microscopy can be combined with contrast enhancing techniques such as DIC illumination to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC.

  • Fluorescence and Phase Contrast Combination Microscopy

    To minimize photobleaching, fluorescence microscopy can be combined with phase contrast illumination. The idea is to locate the specific area of interest in a specimen using the technique (phase) then, without relocating the specimen, switch the microscope to fluorescence mode.

  • Fluorescence Microscopy Digital Image Gallery

    Specimens featured in this gallery contain a variety of discipline examples using specific fluorochrome stains and autofluorescence. Images were captured with either digital camera systems or classical photomicrography on film with Fujichrome Provia 35 millimeter transparency film.

  • Fluorescence Microscopy of Cells in Culture Digital Image Gallery

    The featured fluorescence image gallery explores over 30 of the most common cell lines, labeled with a variety of fluorophores using both traditional staining methods as well as immunofluorescence techniques.

Anatomy of the Fluorescence Microscope

  • Olympus BX51 Upright Microscope

    Learn about the Olympus BX51 Fluorescence Microscope which is an upright epi-fluorescence microscope equipped with a vertical illuminator that contains a turret of filter cubes and a mercury or xenon arc lamp housing.

  • Olympus IX70 Inverted Microscope

    Microscopes with an inverted-style frame (Olympus IX70 Inverted Microscope) are designed for tissue culture applications and are capable of producing fluorescence illumination through an episcopic and optical pathway.

Interactive Java Tutorials

  • Fluorescence Microscope Light Pathways

    Explore illumination pathways in the Olympus BX51 research-level upright microscope. The microscope drawing presented in the tutorial illustrates a cut-away diagram of the Olympus BX51 microscope.

  • Inverted Microscope Light Pathways

    Explore light pathways (in the Olympus IX70) through an inverted tissue culture microscope equipped with for both diascopic (tungsten-halogen) and epi-fluorescence (mercury arc) illumination.

Selected Literature References

Selected Literature References

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

Daniel Axelrod - Department of Biophysics, 930 North University Ave., University of Michigan, Ann Arbor, Michigan 48109.

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.

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, Tennessee, 37212.

Christopher Hardee, Roy Kinoshita, Travis Wakefield, and Robert Johnson - Omega Optical, Inc., 210 Main Street, Brattleboro, Vermont, 05301.

Turan Erdogan - Semrock, Inc., 3625 Buffalo Road, Rochester, New York, 14624.

Mortimer Abramowitz, William K. Fester, Yoshihiro Kawano, and Reinhard G. Enders - Olympus America, Inc., Two Corporate Center Drive, Melville, New York, 11747.

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

Matthew J. Parry-Hill, Thomas J. Fellers, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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