Two-photon excitation microscopy excites fluorescence through the nearly simultaneous absorption of two photons. In the weak unsaturated regime, the excitation rate is proportional to the square of the local illumination intensity. Focusing a pulsed laser therefore strongly favors excitation near its focus rather than throughout the illuminated path.
Scanning that focus provides optical sectioning without relying on a detection pinhole to reject all out-of-focus emission. The photons can have lower individual energies than a single excitation photon, but their combined energy must match an allowed transition. High peak intensity, bleaching and phototoxicity remain relevant; longer excitation wavelength alone does not guarantee less damage.[ref]
Two-photon laser scanning fluorescence microscopy by Winfried Denk and others. Abstract: intrinsic three-dimensional localization through simultaneous two-photon absorption
Articles by others on the same topic
Two Photon Microscopy by Nemonic NeuroNex (2019)
Source. Shows a prototype of a two-photon electron microscope on an optical table, and describes it in good detail, well done.