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Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN OPTICS

  • Vol. 33, Iss. 4 — Feb. 1, 1994
  • pp: 662–669

Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity

David W. Piston, Mark S. Kirby, Heping Cheng, W. J. Lederer, and Watt W. Webb  »View Author Affiliations


Applied Optics, Vol. 33, Issue 4, pp. 662-669 (1994)
http://dx.doi.org/10.1364/AO.33.000662


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Abstract

Two-photon excitation of the ultraviolet-absorbing fluorescent calcium indicator Indo-1 in laser scanning microscopy makes possible a quantitative, three-dimensional recording of intracellular free calcium activity ([Ca2+] i ) distributions and dynamics with low background and minimal photobleaching. We have constructed a simple optical system that facilitates collection of the 400–500-nm Indo-1 fluorescence without the use of a confocal spatial filter. Instead of the fluorescence being descanned as is normally required in confocal microscopy, the fluorescence is deflected by a dichroic mirror into a separate detection pathway. Images of [Ca2+] i distributions with three-dimensional submicrometer resolution and 10% precision are obtained at 100-μM Indo-1 concentration and 3-s recording time for 384 × 512 pixels. Data on [Ca2+] i in tumor mast cells and cardiac myocytes illustrate the capabilities of this technique.

© 1994 Optical Society of America

History
Original Manuscript: April 8, 1993
Revised Manuscript: August 2, 1993
Published: February 1, 1994

Citation
David W. Piston, Mark S. Kirby, Heping Cheng, W. J. Lederer, and Watt W. Webb, "Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity," Appl. Opt. 33, 662-669 (1994)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-33-4-662


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