Two photon microscopy is a new technology that combines laser scanning confocal microscopy and two-photon excitation. The basic principle of two photon microscopy excitation is: In the case of high photon density, the fluorescent molecules absorb two long-wavelength photons at the same time, after a short period of so-called excited state life, a photon with a shorter wavelength is emitted, its effect is the same as using a photon with a wavelength of one-half of a long wavelength to excite the fluorescent molecule. Two-photon excitation requires high photon density, in order to not damage cells, two-photon microscopy usually uses high energy mode-locked pulsed lasers (e.g. Ti sapphire femtosecond laser).
Two photon microscopy has many advantages: 1) Has a strong penetration, two photon microscopy uses long wavelength excitation to easily penetrate specimens, depth of penetration is usually 2 to 3 times that of confocal microscopy; 2) little effect on photobleaching and phototoxicity of the sample, two photon microscopy has photobleaching and phototoxicity only at the focal plane; 3) with high brightness and signal to noise ratio, therefore, two photon microscopy is suitable for long-term observation of living cells and tissues, suitable for deep research on thick biological samples.