The Development of Fluorescence Microscopy

Barry R Masters, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

Published online: January 2010

DOI: 10.1002/9780470015902.a0022093

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.

close

References

Abbe E (1873) Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für mikroskopische Anatomie 9: 413–468.

Alivisatos AP, Gu W and Larabell C (2005) Quantum dots as cellular probes. Annual Review of Biomedical Engineering 7: 55–76.

Brumberg EM (1959) Fluorescence microscopy of biological objects using light from above. Biophysics 4: 97–104.

Denk W, Strickler JH and Webb WW (1990) Two‐photon laser scanning fluorescence microscopy. Science 248: 73–76.

Duncan MD, Reintjes J and Manuccia TJ (1982) Scanning coherent anti‐Stokes Raman microscope. Optics Letters 7: 350–352.

Elson D, Webb S, Siegel J et al. (2002) Biomedical applications of fluorescence lifetime imaging. Optics and Photonics News 13(11): 26–32.

Freudiger CW, Min W, Saar BG et al. (2008) Label‐free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322: 1857–1861.

Girkin J (2008) Laser sources for nonlinear microscopy. In: Masters BR and So PTC (eds) Handbook of Biomedical Nonlinear Optical Microscopy, pp. 191–216. New York: Oxford University Press.

Göppert‐Mayer M (1931) Über Elementarakte mit zwei Quantensprügen. Annalen der Physik 9: 273–294.

Haitinger M (1959) Fluoreszenz‐Mikroskopie, 2. Erweiterte Auflage. Leipzig: Akademische Verlagsgesellschaft. The first edition was published in 1938.

Hell SW and Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated‐emission‐depletion fluorescence microscopy. Optics Letters 19: 780–782.

Huang B, Bates M and Zhuang X (2009) Super‐resolution fluorescence microscopy. Annual Review of Biochemistry 78: 993–1016.

Jablonski A (1935) Über den Mechanisms des Photolumineszenz von Farbstoffphosphoren. Zeitschrift für Physica 94: 38–46.

Janesick JR (2001) Scientific Charge‐Coupled Devices. Bellingham: SPIE Press.

Kasten FH (1983) The development of fluorescence microscopy up through World War II. In: Clark G and Kasten FH (eds) History of Staining, 3rd edn, pp. 147–185. Baltimore: Williams & Wilkins.

Kasten FH (1989) The origins of modern fluorescence microscopy and fluorescent probes. In: Kohen E and Hirschberg JG (eds) Cell Structure and Function by Microspectrofluorometry, pp. 3–50. San Diego: Academic Press.

Kasten FH (1991) Unethical Nazi medicine in annexed Alsace‐Lorraine: the strange case of Nazi anatomist professor Dr. August Hirt. In: Kent GO (ed.) Historians and Archivists, Essays in Modern German History and Archival Policy. Fairfax, VA: George Mason University Press.

Livet J, Weissman TA, Kang H et al. (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450: 56–62.

Masters BR (1996) Selected Papers on Confocal Microscopy, Milestone Series, MS131. Bellingham, WA: SPIE Optical Engineering Press.

Masters BR (2000) The scientific life of Maria Göppert‐Mayer. Optics and Photonics News 11: 38–41.

Masters BR (2003) Selected Papers on Multiphoton Excitation Microscopy, Milestone Series, MS175. Bellingham, WA: SPIE Optical Engineering Press.

Masters BR (2006) Confocal Microscopy and Multiphoton Excitation Microscopy: The Genesis of Live Cell Imaging. Bellingham, WA: SPIE Optical Engineering Press.

Masters BR (2007) Ernst Abbe and the Foundation of Scientific Microscopes. Optics and Photonics News 18: 18–23. Optical Society of America, Washington, DC.

Masters BR (2008a) English translation of: Göppert‐Mayer M (1931). Über Elementarakte mit zwei Quantensprügen, Annalen der Physik (Leipzig), 9: 273–294. In: Masters BR and So PTC (eds) Handbook of Biomedical Nonlinear Optical Microscopy, pp. 44–84. New York: Oxford University Press.

Masters BR (2008b) Cellular metabolism monitored by NAD(P)H imaging with two‐photon excitation microscopy. In: Masters BR and So PTC (eds) Handbook of Biomedical Nonlinear Optical Microscopy, pp. 825–844. New York: Oxford University Press.

Masters BR and So PTC (2008) Handbook of Biomedical Nonlinear Optical Microscopy. New York: Oxford University Press.

McGucken W (1969) Ninteenth‐Century Spectroscopy, Development of the Understanding of Spectra 1802–1897. Baltimore: The Johns Hopkins Press.

Nägerl UV, Willig KI, Hein B, Hell SW and Bonhoeffer T (2008) Live‐cell imaging of dendritic spines by STED microscopy. PNAS 105(48): 18982–18987.

Perinetti G, Müller T, Spaar A et al. (2009) Correlation of 4 Pi and electron microscopy to study transpert through single Golgi stacks in living cells with super resolution. Traffic 10: 379–391.

Ploem JS (1967) The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incidental light. Zeitschrift für Wissenschaftliche Mikroskopie und Mikroskopische Technik 68: 129–142.

Ploem JS and Tanke HJ (1987) Introduction to Fluorescence Microscopy. London: Oxford University Press.

Pringsheim P (1928) Fluorescenz und Phosphorescenz im Lichte Der Neueren Atomtheorie. Berlin: Verlag Von Julius Springer.

Sheppard CJR (1978) The scanning optical microscope. Physics Teacher 16: 648–651.

Sheppard CJR and Kompfner R (1978) Resonant scanning optical microscope. Applied Optics 17: 2879–2882.

Shtengel G, Galbraith JA, Galbraith CG et al. (2009) Interferometric fluorescent super‐resolution microscopy resolves 3D cellular ultrastructure. Proceedings of the National Academy of Sciences of the USA 106: 3125–3130.

Stokes GG (1852) On the change of refrangibility of light. Phil Trans R Soc (London) 142: 463–562.

Tsien RY (1998) The green fluorescent protein. Annual Review of Biochemistry 67: 509–544.

Yazdanfar S and So PTC (2008) Signal detection and processing nonlinear optical microscopes. In: Masters BR and So PTC (eds) Handbook of Biomedical Nonlinear Optical Microscopy, pp. 283–310. New York: Oxford University Press.

Zumbusch A, Holtom GR and Xie XS (1999) Three‐dimensional vibrational imaging by coherent anti‐Stokes Raman scattering. Physical Review Letters 82: 4142–4145.

Further Reading

Davidovits P and Egger MD (1971) Scanning laser microscope for biological investigations. Applied Optics 10: 1615–1619.

De Ment J (1945) Fluorochemistry, A Comprehensive Study Embracing the Theory and Applications of Luminescence and Radiation in Physiochemical Science. New York: Chemical Publishing Company, Inc.

Lakowicz JR (2006) Principles of Fluorescence Spectroscopy, 3rd edn. New York: Springer.

Lockyer JN (1904) Studies in Spectrum Analysis. London: Kegan Paul, Trench, Trübner & Co Ltd.

MacMunn CA (1914) Spectrum Analysis Applied to Biology and Medicine. London: Longmans, Green and Co.

Mycek M‐A and Pogue BW (2003) Handbook of Biomedical Fluorescence. New York: Marcel Dekker, Inc.

Sandison DR and Webb WW (1994) Background rejection and signal‐to‐noise optimization in confocal and alternative fluorescence microscopes. Applied Optics 33: 603–615.

Slomba AF, Wasserman DE, Kaufman GI and Nester JF (1972) A laser flying spot scanner for use in automated fluorescence antibody instrumentation. Journal of the Association for the Advancement of Medical Instrumentation 6: 230–234.

Valeur B (2002) Molecular Fluorescence, Principles and Applications. Weinheim: Wiley‐VCH.

White JG, Amos WB and Fordham M. (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. Journal of Cell Biology 105: 41–48.

Related Sub-Topics:

History | Optical Microscopy
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: The Development of Fluorescence Microscopy
 
How to Cite close
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]