Photon emission by a molecule (fluorescence) occurs in molecules at a high-energy state . A molecule is raised into an excited high energy state when it absorbs a photon of energy equal to the difference between the ground state and the excited state. However, transition into a high-energy state is also possible when a molecule absorbs several low-energy photons at once. While the probability of this event is proportional to the squared value of the intensity of light focused on a sample, such simultaneous absorption and, correspondingly, simultaneous emission (two-photon fluorescence) may only be observed in the focal plane of a microscope system.

The high degree of localisisation of excitation and the use of lower-energy photons compared to conventional fluorescence-based techniques (see fluorescence microscopy) make it possible to image samples at a high focal depth (up to 1 mm) with a high contrast image, protecting the specimens from fading or decomposition due to light effects. Confocal microscopy offers similar opportunities.