Abstract

Confocal laser scanning microscopy (CLSM) provides a significant improvement in axial resolution over conventional epi-fluoeescence microscopy by eliminating out-of-focus fluorescence.¹ This is achieved by using a spatial filter in the form of a confocal aperture. Denk et al. (1990), for the first time, used two-photon-induced fluorescence in conjunction with laser scanning fluorescence microscopy (LSM).² Because the fluorescence quantum efficiency of a two-photon process is quadratically dependent on the illumination intensity, it is possible to achieve depth discrimination even without a confocal aperture in front of the photodetector. Photobleaching in this case occurs only in the viscinity of the focal point. The background scattered light and autofluorescence of the sample in the two-photon-excited system is also lower. The use of IR leads to a larger penetration depth in materials, providing an opportunity to image thicker samples. Furthermore, it is possible to open up the entire visible spectrum for multiple detection channels.

© 1996 Optical Society of America