Two-photon excitation in scanning laser microscopy (Abstract Only)

James H. Strickler; Watt W. Webb

doi:10.1117/12.134895

1 February 1992 Two-photon excitation in scanning laser microscopy (Abstract Only)

James H. Strickler, Watt W. Webb

Proceedings Volume 1556, Scanning Microscopy Instrumentation; (1992) https://doi.org/10.1117/12.134895
Event: San Diego, '91, 1991, San Diego, CA, United States

Abstract

Two-photon excitation in scanning laser fluorescence microscopy provides axial resolution and rejection of out of focus background similar to that provided by confocal microscopy. Two- photon excitation is allowed by the extremely high peak intensity (> 1010 W/cm2) at the waist of a highly focused beam of subpicosecond pulses from a modelocked laser. Chromophore excitation and photobleaching are restricted to the focal plane by quadratic dependence of the excitation rate on incident intensity. Sectional imaging is therefore possible without the need of a confocal aperture allowing the use of UV excited chromophores, including quantitative fluorescent indicators of divalent cations, which are problematic for confocal microscopy because of chromatic aberration of objective lenses. Furthermore, sectional imaging becomes possible using widefield viewing detectors such as CCD arrays or video camera. The confinement of two-photon excitation to the focal volume allows point localized photochemistry. Two-photon excited release of caged biological probes makes possible the observation of the living cell's response to chemical stimuli with the spatial resolution of optical microscopy. Two-photon excited laser scanning photolithography allows the generation of complicated 3-D forms with a single patterned exposure and one development step. Two-photon activation of fluorescent dyes allows 3-D optical data storage at extremely high density. Two-photon excited release of caged fluorescent probes allows micromobility measurements in 3-D bulk media. By photolytic release of fluorescent dye and subsequent observation of its redistribution it is possible to quantitatively study diffusional transport properties in polymers and living biological specimens.

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James H. Strickler and Watt W. Webb "Two-photon excitation in scanning laser microscopy (Abstract Only)", Proc. SPIE 1556, Scanning Microscopy Instrumentation, (1 February 1992); https://doi.org/10.1117/12.134895

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KEYWORDS

Confocal microscopy

Microscopy

Chromophores

3D metrology

CCD image sensors

Lenses

Luminescence

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