The classic method of nonlinear absorption used by microscopists is conventional two-photon fluorescence, in which two photons from a single source interact to excite a photoelectron. The electron then emits a photon as it transitions back to its ground state. This microscopy method has been revolutionary in biological sciences because of its inherent three-dimensional optical sectioning capabilities.
Two-photon absorption is inherently a nonlinear process: fluorescent output intensity is proportional to the square of the excitation light intensity. This ensures that fluorescence is only generated within the focus of a laser beam, as the intensity outside of this plane is insufficient to excite a photoelectron.
However, this microscope modality is inherently limited by the number of biological molecules that can undergo both two-photon excitation and fluorescence.
Pump–probe microscopy circumvents this limitation by directly measuring excitation light. This expands the number of potential targets to any molecule capable of two-photon absorption, even if it does not fluoresce upon relaxation. The method modulates the amplitude of a pulsed laser beam, referred to as the pump, to bring the target molecule to an excited state. This will then affect the properties of a second coherent beam, referred to as the probe, based on the interaction of the two beams with the molecule. These properties are then measured by a detector to form an image.