Basic Principles of Multiphoton Excitation Microscopy

Multiphoton microscopy is one of the fastest growing areas in biomedical imaging. The potential of multiphoton excitation was first theorized by Maria GoppertMayer in 1931. Generating three-dimensionally resolved microscopic images based on nonlinear optical excitation was postulated in the 1970s (Gannaway and Sheppard, 1978; Wilson and Sheppard, 1984). The definitive experiment was done by Denk, Webb, and co-workers (1990), who accomplished two-photon, three-dimensional (3D) imaging of biological specimens. Furthermore, they demonstrated 3D localized uncaging and photobleaching using two-photon excitation to trigger a photochemical reaction in a subfemtoliter volume.