The three-photon (3P) microscopy allows noninvasive functional imaging by making cells visible also in the deeper tissues with high spatial-resolution and better contrast compared to the two-photon excitation. The optical design required for 3P excitation establishes higher axial resolution than that of the two-photon microscopy. The spectral window enables three-photon excitation of a variety of fluorophores, such as the current generations of protein-based genetically encoded calcium indicators (e.g. GCaMP6) and the repetition rate of the laser source is adequate for imaging Ca2+ transients produced from neural activity.