is 1.3 with an
oil immersion objective. Two-photon excitation employs
two-photon absorption, a concept first described by
Maria Goeppert Mayer (1906–1972) in her doctoral dissertation in 1931, and first observed in 1961 in a CaF2:Eu2+ crystal using
laser excitation by
Wolfgang Kaiser.
Isaac Abella showed in 1962 in
caesium vapor that two-photon excitation of single atoms is possible. Two-photon excitation fluorescence microscopy has similarities to other confocal laser microscopy techniques such as
laser scanning confocal microscopy and
Raman microscopy. These techniques use focused laser beams scanned in a raster pattern to generate images, and both have an
optical sectioning effect. Unlike confocal microscopes, multiphoton microscopes do not contain pinhole apertures that give confocal microscopes their optical sectioning quality. The optical sectioning produced by multiphoton microscopes is a result of the
point spread function of the excitation. The concept of two-photon excitation is based on the idea that two photons, of comparably lower photon energy than needed for one-photon excitation, can also excite a
fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. The emitted photon is at a higher energy (shorter wavelength) than either of the two exciting photons. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore, a high peak
flux of excitation photons is typically required, usually generated by femtosecond
pulsed laser. For example, the same average laser power but without pulsing results in no detectable fluorescence compared to fluorescence generated by the pulsed laser via the two-photon effect. The longer wavelength, lower energy (typically infrared) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than the short-wavelength lasers typically used for single-photon excitation, so living tissues may be observed for longer periods with fewer toxic effects. The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the two-photon fluorescence lies in the ~700–1100 nm (infrared) range produced by
Ti-sapphire lasers. If the fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to be raised into the excited state. The fluorophore will then emit a single photon with a wavelength that depends on the type of fluorophore used (typically in the
visible spectrum). Because two photons are absorbed during the excitation of the fluorophore, the probability of fluorescent emission from the fluorophores increases quadratically with the excitation intensity. Therefore, much more two-photon fluorescence is generated where the laser beam is tightly focused than where it is more diffuse. Effectively, excitation is restricted to the tiny focal volume (~1 femtoliter), resulting in a high degree of rejection of out-of-focus objects. This
localization of excitation is the key advantage compared to
single-photon excitation microscopes, which need to employ elements such as pinholes to reject out-of-focus fluorescence. The fluorescence from the sample is then collected by a high-sensitivity detector, such as a
photomultiplier tube. This observed light intensity becomes one
pixel in the eventual image; the focal point is scanned throughout a desired region of the sample to form all the pixels of the image. ==Development==