ABSTRACT
Conventional fluorescence microscopy was revolutionized by the sectioning ability provided by the confocal microscope. As a rule of thumb for most fluorochromes, the optimal two-photon excitation wavelength will be slightly less than twice the wavelength required for single-photon excitation. In addition, the two-photon excitation spectrum is significantly broadened and blue-shifted compared to a doubled one-photon spectrum. Microscope optics generally perform poorly in the near-UV region since the rapid change in refractive index in this region for most glasses leads to chromatic aberrations. The spatial restriction of two-photon excitation also limits inner filtering effects due to absorption of the excitation light as it passes through the specimen. In addition the UV light normally used for uncaging is damaging to biological specimens; it passes throughout the specimen causing damage everywhere. Since the aim of such experiments is to examine dye movement in living cells, this is a serious limitation. Long-wavelength light is much less damaging to living cells than ultraviolet light.
