Two-photon absorption (TPA) is a third-order process in which two photons are nearly simultaneously absorbed by the same molecule. If a second photon is absorbed by the same electron within the same quantum event, the electron enters an excited state.
This is the same phenomenon used in two-photon microscopy (TPM), but there are two key features that distinguish pump–probe microscopy from TPM. First, since the molecule is not necessarily fluorescent, a photodetector measures the probe intensity. Therefore, the signal decreases as two-photon absorption occurs, the reverse of TPM.
Second, pump–probe microscopy uses spectrally separated sources for each photon, whereas conventional TPM uses one source of a single wavelength. This is referred to as degenerate two-photon excitation.