Fig. 1.

Principles of 2PE microscopy. (a) Jablonski energy diagram showing the electron excitation process in single- (left) and two-photon (right) fluorescence microscopy. Single-photon excitation (left) requires the absorption of a high energy photon to excite an orbital electron of a fluorophore to a vibrationally and electronically excited state (excitation). When this electron relaxes to its ground state, it emits a photon of light of a different wavelength than the excitation photon (fluorescence emission). In 2-photon excitation (2PE; right), this process is achieved by the quasi-simultaneous absorption of two photons; the first one excites the electron to a virtual intermediate state, and the second one completes the excitation to reach the electronic excited state. (b) Confocal versus 2PE microscope systems. In confocal systems (left), the presence of a pinhole right before the photodetector rejects the photons emitted from outside the focus (e.g., photon #6), as well as those scattered on their way to the PMT (e.g., photon #4). Only unscattered photons coming from the focal plane are able to pass through the pinhole (e.g., photon #5) and contribute to the signal. In 2PE systems (right) there is no need for a pinhole since photons contributing to the signal come only from the geometrical focus of the excitation spot (e.g., photon #5), even if they were scattered on their path to the PMT (e.g., photon #4)