Time-resolved fluorescence imaging and background rejection by two-photon excitation in laser-scanning microscopy

David W. Piston; David R. Sandison; Watt W. Webb

doi:10.1117/12.58230

1 April 1992 Time-resolved fluorescence imaging and background rejection by two-photon excitation in laser-scanning microscopy

David W. Piston, David R. Sandison, Watt W. Webb

Author Affiliations +

Proceedings Volume 1640, Time-Resolved Laser Spectroscopy in Biochemistry III; (1992) https://doi.org/10.1117/12.58230
Event: OE/LASE '92, 1992, Los Angeles, CA, United States

Abstract

A new dimension in quantitative fluorescence microscopy may be accessed by imaging of fluorescence decay times. To obtain spatially resolved information from microscopic sample locations, one must not only have sufficient optical resolution and detection sensitivity but also the ability to exclude fluorescence photons originating from outside the focal volume of interest. This background rejection is measured by the signal-to-background ratio, which must be large if three-dimensional information is to be obtained from a thick fluorescence sample. Two-photon excitation in laser scanning microscopy has an unparalleled ability to meet these demands. The two-photon excitation of a transition normally in the ultraviolet arises from the simultaneous non-linear absorption of two red photons. Because two-photon excitation depends on the square of the incident intensity, the resulting fluorescence is limited to the focal volume where the photon density of the focused laser illumination is high. This localization limits photobleaching and any photodamage to the focal plane of the image. This property is a major advantage over widefield or confocal microscopy. Two-photon excitation provides the depth discrimination associated with confocal microscopy without a confocal spatial filter, an advantage which allows for major simplifications of the apparatus. The resolution and background rejection properties of two-photon excitation have been calculated and measured, and have been shown to be identical to an ideal confocal microscope with the same optical wavelengths. Combined, these properties provide the ideal conditions for time-resolved imaging of fluorescence decay dynamics in order to characterize the submicroscopic environment of the fluorophore molecules within the specimen. A preliminary apparatus was designed and built to test these concepts, and it was found that fluorescence decay time images of living cells can be conveniently recorded with diffraction limited resolution in a few seconds of image acquisition time.

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David W. Piston, David R. Sandison, and Watt W. Webb "Time-resolved fluorescence imaging and background rejection by two-photon excitation in laser-scanning microscopy", Proc. SPIE 1640, Time-Resolved Laser Spectroscopy in Biochemistry III, (1 April 1992); https://doi.org/10.1117/12.58230

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