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

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.