The Development of Fluorescence Microscopy

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

Figure 1.

Slit ultramicroscope devised by Siedentopf and Zsigmondy (1902). Reproduced with permission from Carl Zeiss archives.

Figure 2.

Slit ultramicroscope devised by Siedentopf and Zsigmondy (1923) (a) to observe ultramicroscopic particles in solid bodies and (b) to observe ultramicroscopic flowing particles. Reproduced with permission from Carl Zeiss archives.

Figure 3.

Slit ultramicroscope devised by Siedentopf and Zsigmondy (1930) with a cuvette to investigate ultramicroscopic flowing particles. Reproduced with permission from Carl Zeiss archives.

Figure 4.

Luminescence microscope developed by Zeiss/Jena after design of Ellinger–Hirt. It used incident illumination to observe biological specimens in intravital fluorochroming. Reproduced with permission from Carl Zeiss archives.

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Masters, Barry R(Jan 2010) The Development of Fluorescence Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022093]