Two-Photon Excitation microscopy is based on the nonlinear optical excitation of a fluorophore induced by a high peak power of ultrafast laser pulses. In contrast to the classical fluorescence microscopy, where the exciting photon has higher energy than the emitted one, 2PE is induced in the fluorescent molecule by two exciting photons with a wavelength longer than the one of the emitted light.

The strength of this process lies in the fact that it allows a 3D sectioning of the sample without the need of a confocal pinhole, since the fluorescence is restricted to a small focal volume. 2PE-FLIM can combine the advantage of both techniques, allowing imaging of thick live samples and monitoring the lifetime of fluorescent molecules at the same time.