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Proceedings Paper

Two-photon excitation in laser scanning fluorescence microscopy
Author(s): James H. Strickler; Watt W. Webb
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Paper Abstract

Simultaneous absorption of two red photons from a strongly focused subpicosecond colliding pulse mode4ocked dye laser stimulates visible fluorescence emission from fluorophores having their normal absorption in the ultraviolet1. The quadratic increase of the two-photon excitation rate with excitation intensity restricts fluorescence emission to the focal volume thus providing the same depth resolution as does confocal microscopy. Image degradation due to out of focus backround is thus avoided. Photobleaching and most cellular photodamage are similarly confined to the focus thereby minimizing sample degredation during acquisition of the multiple sections required for 3-d image reconstruction. Fluorescence images of living cells and other thick photolabile fluorescence labled assemblies illustrate the depth discrimination of both two-photon fluorescence excitation and photobleaching. The quadratic intensity dependence of two-photon excitation allows 3-d spatially resolved photochemistry in particular the photolytic release of caged compounds such as neurotransmitters nucleotides fluorescent dyes and second messengers such as 1P3 and Ca. The two-photon release of cased ATP has been measured and release of a caged fluorescent dye has been shown. Point photobleaching and a 3-d " write once read many" optical memory have been demonstrated. Two-photon excitation of photo-initiated polymerization with a sharply focused single beam allows microfabrication of complex structures of arbitrary form. By scanning the focused beam through a liquid polymer with a UV excited initiator it is possible to harden the polymer only at the focus thereby creating

Paper Details

Date Published: 1 April 1991
PDF: 12 pages
Proc. SPIE 1398, CAN-AM Eastern '90, (1 April 1991); doi: 10.1117/12.47787
Show Author Affiliations
James H. Strickler, Cornell Univ. (United States)
Watt W. Webb, Cornell Univ. (United States)

Published in SPIE Proceedings Vol. 1398:
CAN-AM Eastern '90

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