Two-photon Fluorescence Light Microscopy

The invention of two-photon fluorescence light microscopy by Denk, Webb and co-workers (Denk et al., 1990) revolutionized three-dimensional (3D) in vivo imaging of cells and tissues. In 1931, the theoretical basis of twophoton excitation was established by Maria GöppertMayer, and this photophysical effect was verified experimentally by Kaiser and Garret in 1963. Two-photon excitation is a fluorescence process in which a fluorophore (a molecule that fluoresces) is excited by the simultaneous absorption of two photons (Figure 1). The familiar onephoton fluorescence process involves exciting a fluorophore from the electronic ground state to an excited state by a single photon. This process typically requires photons in the ultraviolet or blue/green spectral range. However, the same excitation process can be generated by the simultaneous absorption of two less energetic photons (typically in the infrared spectral range) under sufficiently intense laser illumination. This nonlinear process can occur if the sumof the energies of the twophotons is greater than the energy gap between the molecule’s ground and excited states. Since this process depends on the simultaneous absorption of two infrared photons, the probability of two-photon absorption by a fluorescent molecule is a quadratic function of the excitation radiance. Under sufficiently intense excitation, three-photon and higherphoton excitation is also possible and deepUVmicroscopy based on these processes has been developed. Article