Multiphoton microscopy

Two-photon or multi-photon excitation microscopy (MP) is a fluorescence imaging technique that, similar to confocal microcopy, eliminates out-of-focus light resulting in very sharp images. This is achieved through the use of a pulsed laser, unlike the continuous emission lasers used in m. conventional confocal, emitting in infrared wavelengths (> 700 nm). For the detection of fluorescence, you can use the confocal microscope in which it is installed although, in some cases, it is necessary to use detectors coupled directly to the stand (direct detectors), which collect most of the light thus improving the sensitivity. The physical principle of this technique is that fluorescent molecules absorb light of greater wavelength when they simultaneously absorb 2 or more photons, an event that only takes place in the focal plane of the sample. Therefore, the backlight (out of focus) is strongly suppressed and makes unnecessary the use of an iris-diaphragm in the conjugate plane of the image used in conventional confocal. This, together with the low phototoxicity and dispersion of infrared light, make the m. MP a superior alternative to conventional confocal microscopy for deep inspection of tissues, living and/or fixed ones.