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The Development of Fluorescence Microscopy

  1. Barry R Masters

Published Online: 15 JAN 2010

DOI: 10.1002/9780470015902.a0022093

eLS

eLS

How to Cite

Masters, B. R. 2010. The Development of Fluorescence Microscopy. eLS. .

Author Information

  1. Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

Publication History

  1. Published Online: 15 JAN 2010

Abstract

The fluorescence microscope (wide-field, scanning, confocal, one-photon excitation, multiphoton excitation) is an extremely useful and ubiquitous instrument in biological and medical laboratories. The fluorescence microscope provides enhanced contrast, single protein specificity and single molecule sensitivity. Progress in the technical development over more than 100 years of instrument design and fluorescent probe synthesis has contributed to the continuing widespread utility of the fluorescence microscope. Modern advances in instrumentation include the use of continuous wave and pulsed laser light sources, dichroic filters, photomultipliers, avalanche photodetectors and charge-coupled device detectors. The invention of the two-photon excitation microscope provides for microscopic imaging of live cells and whole organisms. The development of specific dyes, physiological probes and molecular probes (intrinsic and extrinsic) stimulated the widespread use of fluorescence microscopy in the life sciences. Recently, genetically expressed fluorescent proteins and quantum dots provide new research capabilities for the intravital fluorescence microscope.

Key concepts:

  • The reader will gain an understanding of the development of both the fluorescence microscope instrumentation and the field of fluorescent probe synthesis, development, limitations and applications to the life sciences.

  • The fluorescence microscope can provide single molecule detection sensitivity.

  • The fluorescence microscope can provide single protein selectivity.

  • A major advance in fluorescence microscope is the development of multiphoton excitation microscopy.

  • Recent instrument advances include the following: laser light sources, dichroic mirror filters, different types of photodetectors and imaging cameras.

  • Recent advances in fluorescent probe development include the following: genetically expressed fluorescent proteins and quantum dots.

  • The limitations of the fluorescence microscope include the following: optical resolution, noise and aberrations; photon-induced cell and tissue damage; the bleaching of fluorescent probes.

  • There are microscopic techniques that do not depend on fluorescent probes, but have alternative contrast mechanisms, that is, coherent anti-Stokes Raman microscopy and second-harmonic generation microscopy.

Keywords:

  • fluorescence microscope;
  • fluorescence;
  • fluorescence molecular probes;
  • genetically expressed fluorescent proteins;
  • quantum dots