ABSTRACT
Fluorescence is normally the result of absorption of an individual photon of energy by a fluorescent molecule and the subsequent re-emission of some of that absorbed energy as light. For each fluorescent molecule, this process is repeated multiple times, until excited state reactions completely bleach the fluorophore. Multiphoton absorption is the simultaneous (vs. sequential) absorption of multiple photons in a single quantized event that combines their excitation energies to produce an electronic excitation that is conventionally caused by a single photon of a shorter wavelength. Unlike single photon absorption, where the absorption efficiency is independent of the time of arrival of the exciting photon, multiphoton absorption probability is dictated by both spatial and temporal overlap of the incident photons. Confocal imaging systems remove out-of-focus information by illuminating a single point of the specimen with a focused beam, and by placing a pin-hole aperture in the emission light pathway at a focal plane conjugate to the specimen.
