Multi-Photon Microscopy (MPM)

Multi-photon microscopy (MPM) has become a well established optical imaging technique since its first exploitation more than a decade ago. MPM relies on a nonlinear optical technique whereby two or more photons may be directly absorbed by a fluorescent molecule, causing the emission of a photon at a shorter wavelength (see figure below). Since it is a nonlinear effect, it is highly dependent on peak incident intensity and tight focusing. In order to illuminate the sample with the necessary high peak intensity, while preventing thermal damage, a femtosecond pulsed laser source is commonly used. This laser source is then focused through a micrsocope objective into a sample treated with fluorescent dye.

The photons of the laser source do not, individually, have enough energy to cause fluorescence. It is only in the center of a tightly focused beam that the photon density is high enough to cause direct absorption of two photons, resulting in an up-converted emission. This means that a fluorescence signal will only be detected from the focus of the laser beam. The focus can then be rasterized through the sample to create a 2D image or a 3D volume, in much the same way as confocal microscropy.