Fluorescence Microscopy

Abstract

Fluorescence microscopy in biomedical research is a light microscope technique designed to view fluorescence emission from a biological specimen. It has become an extremely useful tool to localize genes, messenger ribonucleic acid (mRNA) and proteins within live and fixed cells and tissues. Cell biology has been revolutionized by the discovery and applications of green fluorescent protein, and its many mutant forms, that can be fused to a cellular protein of interest, making it possible to localize and study the dynamics of that protein in living cells. In addition, the development of other fluorescent reporters, based on small molecule dyes, has made it possible to visualize spatiotemporal variations in intracellular calcium as well as other ions and metabolites and the activity of components in signal transduction networks using specialized biosensors.

Key Concepts

  • Fluorescence microscopy enables visualization of the emission from fluorescent compounds in various specimens to submicron spatial resolution.

  • Fluorophores are molecules that emit longer wavelength that emit light after absorption of shorter wavelength light.

  • Biosensors are fluorophores that report on the location and activity of molecules or ions that, typically, are involved in signalling networks.

Keywords: fluorescence microscopy; fluorescent probes; biosensors; lasers; immunofluorescence; fluorescence in situ hybridization

Figure 1.

Schematic diagram of the basic fluorescence microscope. —, exciting light and – – –, fluorescent light.

Figure 2.

An example of an immunofluorescence micrograph. Optical section of a metaphase cell showing chromosomes stained with an anti‐DNA antibody (red), centromeres labelled with an anticentromere antibody (green) and the spindle apparatus stained with an antitubulin antibody (purple). The image was taken using a confocal microscope and pseudocoloured. The micrograph was provided by Dr Vasily Ogryzko, National Institutes of Health, and was taken at the Cold Spring Harbor course on ‘Advanced In Situ Hybridization and Immunocytochemistry’, 1995.

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References

Hodgeson L, Pertz O and Hahn KM (2008) Design and optimization of genetically encoded fluorescent biosensors: GTPase biosensors. Methods in Cell Biology 85: 63–81.

Jameson D (1984) Fluorescence principles, methodologies, and applications. In: Voss E Jr (ed.) Fluorescein Hapten: An Immunological Probe, Uniscience Series, pp. 23–48. Boca Raton, FL: CRC Press.

Kramer J (1999) The right filter set gets the most out of a microscope. Biophotonics International 5: 54–59.

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Tsien R and Miyawaki A (1998) Seeing the machinery of live cells. Science 280: 1954–1955.

Tsien R and Waggoner A (1995) Fluorophores for confocal microscopy: photophysics and photochemistry. In: Pawley J (ed.) The Handbook of Biological Confocal Microscopy, pp. 267–277. Madison, WI: IMR Press.

Waters JC (2009) Accuracy and precison in quantitative fluorescence microscopy. Journal of Cell Biology 85: 1135–1148.

Willis RC (1999) Examining live embryos nondestructively. Biophotonics International 6: 42–44.

Further Reading

Lakowicz J (1983) Principles of Fluorescence Spectroscopy. New York: Plenum Press.

Spector DL, Goldman RD and Leinwand LA (1998) Cells: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Sweet D (1999) Frontiers in imaging technology. Trends in Cell Biology 9(Special Issue): 43–77.

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How to Cite close
Jacobson, Ken(Mar 2010) Fluorescence Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002637.pub2]