Multiphoton fluorescence microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation. At high photon densities, two (or more) photons can be simultaneously absorbed by combining their energies to provoke the electronic absorption of a fluorophore in the specimen. This will then result in fluorescence emission in the visible region. Excitation in multiphoton microscopy occurs only at the focal point of a diffraction-limited microscope objective, thus providing the ability to optically section thick biological and other transparent specimens in order to obtain three-dimensional resolution.
Because the energy of a photon is inversely proportional to its wavelength, the two photons should have wavelengths about twice that required for single-photon excitation and the excitation sources need to be emitting in the red or infrared regions. In addition, unlike the case for single-photon absorption, the probability that a given fluorophore will simultaneously absorb two photons is a function of both the spatial and temporal overlap between the incident photons. In fact, photon concentration must be approximately a million times that required for an equivalent number of single-photon absorptions. This is accomplished with high-power mode-locked pulsed lasers, which generate a significant amount of power during pulse peaks. Individual optical sections are acquired by raster scanning the specimen in the x-y plane with the laser beam and detecting the induced fluorescence, and a full three-dimensional image is composed by serially scanning the specimen at sequential z positions. Because the position of the focal point can be accurately determined and controlled, multiphoton fluorescence is useful for probing selected regions beneath the specimen surface. The highly localized excitation energy minimizes photobleaching of fluorophores attached to the specimen and reduces photodamage.
In multiphoton microscopy, photons emitted through fluorescence originate almost exclusively from the objective focal plane, eliminating the requirement for descanned detection and permitting more flexible detection geometries. This increased versatility leads to a considerable improvement in fluorescence detection efficiency.
This text is an adaptation from http://www.olympusmicro.com/primer/techniques/fluorescence/multiphoton/multiphotonintro.html
Olympus' Fluoview FV1000-MPE, is a multiphoton laser scanning microscope that allows fluorescence imaging deep within specimens. Utilizing ultrashort pulsed IR laser, the Olympus Fluoview MPE is able to image hundreds of microns into a specimen thanks to the penetration of IR light and uniquely designed long working distance objectives. The Fluoview-MPE provides cutting-edge technology for various areas of scientific research such as neuroscience and cell biology. Optimal for in-vivo observation requiring deeper imaging.