Fluorescence Microscopy

Fluorescence microscopy, the use of fluorescence illumination and observation, is the most rapidly expanding microscopy technique employed today, both in the medical and biological sciences. Fluorescence microscopy’s prevalence has spurred the development of more sophisticated microscopes and numerous fluorescence accessories. Epifluorescence, 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 microscopy section of the primer into several categories to make it easier to organize and download. Follow the links below to navigate to points of interest relating to fluorescence microscopy.
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Fluorescence Microscopy

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

To generate enough excitation light intensity to provide secondary fluorescence emission capable of detection, powerful light sources are needed such as LED, mercury, and xenon arc (burner) lamps.

Introduction to Laser Scanning Microscopes

Learn all about laser scanning microscopes, including their advantages, disadvantages, and the types of images they can produce.

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.

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 Microscopy Errors

Read more about how 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, who 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.

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.