kahefotonmikroskoopia

Kahefotonmikroskoopia, also known as two-photon excitation microscopy or TPEF, is a confocal microscopy technique that uses non-linear optical effects to excite fluorophores. In this method, two low-energy photons are simultaneously absorbed by a fluorophore to excite it to a higher energy state. The energy of these two photons, when combined, is equivalent to the energy of a single higher-energy photon that would typically be used for excitation in conventional fluorescence microscopy.

A key advantage of kahefotonmikroskoopia is its ability to achieve optical sectioning with reduced photobleaching and photodamage. Because the excitation only occurs at the focal point where the photon density is high enough for the simultaneous absorption to occur, out-of-focus fluorescence is significantly minimized. This allows for deeper penetration into scattering samples, such as biological tissues, compared to traditional confocal microscopy.

The technique typically employs a pulsed infrared laser, which is less damaging to biological samples than visible light lasers. The longer wavelength of infrared light also leads to better penetration through scattering media. Kahefotonmikroskoopia finds widespread applications in neuroscience for imaging neuronal activity in vivo, in developmental biology for observing cellular processes in living organisms, and in materials science for non-destructive imaging of thick specimens. The precise localization of excitation and the reduced scattering enable high-resolution imaging in complex, three-dimensional environments.