Multi-photon confocal microscopy

Multi-photon, or more generally two-photon, microscopy is a point scanning technique that uses a pulsed infrared (IR) laser that has a tunable wavelength.  At a wavelength approximately twice the excitation maximum of a fluorophore, if two photons simultaneously interact with the fluorophore it can be excited.  These events generally are restricted to the exact focal point of the laser, so an extremely small volume is excited and no emission pinhole is required.  Furthermore, as IR light penetrates deep into tissue multi-photon confocal microscopy is an excellent choice for intravital imaging.


Nature Biotechnology 21, 1369–1377 (2003)