Multiphoton (MP) microscopy utilizes a non-linear excitation process, usually two-photon excitation, which occurs only at the focal point of the microscope. This gives inherent optical sectioning capabilities, without cutting off out-of-focus emission, and minimizes the photobleaching and photodamage that are the ultimate limiting factors in imaging live cells. The low energy/ long wavelenght infrared (IR) excitation light is less harmful to living species than the light range used for confocal microscopy. The IR light also undergoes less scattering, which results in less background and longer penetration depths. These advantages allows investigations on thick living tissue specimens that would not otherwise be possible with conventional imaging techniques.
In addition to multiphoton excitation of fluorophores, it is also possible to perform other non-linear microscopy techniques, like second harmonic generation (SHG).