What is 3-photon excitation?
In 3-photon excitation, 3 photons are absorbed by a fluorophore molecule. The fluorophore is promoted to an excited state. The absorbed energy is typically lost through radiationless relaxation before decay to the ground state, accompanied by the emission of a photon.
For GFP, excitation may occur through absorption of a blue photon at ~470 nm (linear or single-photon excitation), of two infrared photons at ~910 nm (2-photon excitation), or of three infrared photons at ~1300 nm (3-photon excitation) and fluorescence emission is in the green spectrum at ~510 nm.
Transitions of the fluorophore between energy states are often illustrated schematically with a Jablonski diagram (Jablonski, 1933).
The absorption of a single photon results in a linear relationship between illumination and fluorescence intensities. 2-photon and 3-photon excitation exhibit quadratic and cubic dependence of fluorescence on illumination intensity, respectively. The intrinsic optical sectioning of 2- and 3-photon excitation results from the non-linear relationship between illumination intensity and fluorescence.