Three-photon microscopy

Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect.^[1][2][3] Different from two-photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons. The fluorescent dyes then emit one photon whose energy is (slightly smaller than) three times the energy of each incident photon. Compared to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plane by ${\displaystyle 1/z^{4}}$, which is much faster than that of two-photon microscopy by ${\displaystyle 1/z^{2}}$.^[4] In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect. This causes three-photon microscopy to have higher resolution than conventional microscopy.