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Two-Photon Molecular Excitation in Laser-Scanning Microscopy

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Handbook of Biological Confocal Microscopy

Abstract

Molecular excitation by two-photon absorption holds great promise for vital imaging of biological systems using laser-scanning microscopy. Fluorescence microscopy, which can provide submi-cron spatial resolution of chemical dynamics within living cells, is frequently limited in its sensitivity and spatial resolution by background due to out-of-focus fluorescence. Two-photon excitation avoids this background in laser-scanning microscopy by virtue of its nonlinear optical absorption character, which almost completely limits the excitation to the high-intensity region at the focal point of the strongly focused excitation laser. Since excitation of background fluorescence is avoided, no confocal spatial filter is required, and we obtain all of the advantages of a linear (one-photon) confocal microscope plus the absence of out-of-focus photobleach-ing and photodamage.

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Author information

Authors and Affiliations

  1. AT & T Bell Laboratories, 600 Mountain Avenue, Room 3L-404, Murray Hill, New Jersey, 07974, USA

    Winfried Denk

  2. Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, Tennessee, 37233, USA

    David W. Piston

  3. Developmental Resource for Biophysical Imaging and Optoelectronics, Cornell University, Ithaca, New York, 14853, USA

    Watt W. Webb

Authors
  1. Winfried Denk
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  2. David W. Piston
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  3. Watt W. Webb
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Editor information

Editors and Affiliations

  1. Zoology Department, University of Wisconsin-Madison, Madison, Wisconsin, USA

    James B. Pawley

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Denk, W., Piston, D.W., Webb, W.W. (1995). Two-Photon Molecular Excitation in Laser-Scanning Microscopy. In: Pawley, J.B. (eds) Handbook of Biological Confocal Microscopy. Springer, Boston, MA. https://doi.org/10.1007/978-1-4757-5348-6_28

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  • DOI: https://doi.org/10.1007/978-1-4757-5348-6_28

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4757-5350-9

  • Online ISBN: 978-1-4757-5348-6

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