Time-Resolved Fluorescence Imaging and Background Rejection by Two-Photon Excitation in Laser Scanning Microscopy

摘要

A new dimension in quantitative fluorescence microscopy can be accessed by imaging of fluorescence decay times. To obtain spatially resolved information from microscopic sample locations, one must have not only 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 unparalleled capability to meet these demands. The two-photon excitation of a transition normally excited by ultraviolet photons arises from the simultaneous non-linear absorption of two red photons. Because the rate of 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 also limits photobleaching and any photodamage to the focal plane of the image, an advantage over both widefield and confocal microscopy. Two-photon excitation provides depth discrimination matching confocal microscopy without requiring confocal spatial filter; this advantage allows 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 ideal conditions for time-resolved imaging of fluorescence decay dynamics in order to characterize the submicroscopic environment of the fluorophore molecules within the specimen. We have designed and built a preliminary apparatus to test these concepts and have 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.