Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. It produces three-dimensional and deep tissue imaging. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Two-photon excitation microscopy is used, in preference to confocal microscopy, for experiments that require large image depths in live tissue or in small animals.