Two-photon excitation microscopy

The two-photon excitation (TPE) microscopy is the most relevant advancement in fluorescence optical microscopy and has been strongly influenced by the availability of ultrafast pulsed lasers. It offers real progress in science with its intrinsic three-dimensional resolution. The absence of background fluorescence, and the attractive possibility of exciting UV excitable fluorescent molecules increases the sample penetration. In a TPE scheme two 720-nm photons combine to produce the same fluorescence conventionally primed at 360 nm. TPE process reduces phototoxicity and is advantageous compared with the damage induced by means of conventional fluorescence excitation. It has been found that there is no photon signal from sources out of the geometric position of the optical focus and thus the signal-to-noise ratio increases. it is also observed that the fluorescence emission occurs at a wavelength shorter and at a larger spectral distance than in the one-photon excitation.