two-photon microscopy (rus. микроскопия, двухфотонная) — a technique used for detecting fluorescent microscale objects using a light microscope, where the object's fluorescence is induced as a result of simultaneous absorption of two photons of comparable lower energy than needed for fluorescence excitation.


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.


  • Borisenko Grigory G.


  1. Denk W., Strickler J.H., Webb W.W. Two-photon laser scanning fluorescence microscopy // Science. 1990. V. 248. P. 73–76.

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