Two-photon excitation microscopy
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging living tissue up to a depth of one millimeter. The two-photon excitation microscope is a special variant of the multiphoton fluorescence microscope. Two-photon excitation may in some cases be a viable alternative to confocal microscopy due to its deeper tissue penetration and reduced phototoxicity.
Two-photon excitation employs a concept first described by Maria Goppert-Mayer (b. 1906) in her 1931 doctoral dissertation.
The concept of two-photon excitation is based on the idea that two photons of low energy can excite a fluorophore in a quantum event, resulting in the emission of a fluorescence photon, typically at a higher energy than either of the two excitatory photons. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore a high flux of excitation photons is typically required, usually a femtosecond laser.
Two-photon microscopy was pioneered by Winfried Denk in the lab of Watt W. Webb at Cornell University. He combined the idea of two-photon absorption with the use of a laser scanner. In two-photon excitation microscopy an infrared laser beam is focused through an objective lens. The Ti-sapphire laser normally used has a pulse width of approximately 100 femtoseconds and a repetition rate of about 80 MHz, allowing the high photon density and flux required for two photons absorption and is tunable across a wide range of wavelengths. Two-photon technology is patented by Winfried Denk, James Strickler and Watt Webb at Cornell University.
The most commonly used fluorophores have excitation spectra in the 400500 nm range, whereas the laser used to excite the fluorophores lies in the ~7001000 nm (infrared) range. 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 need to be absorbed to excite a fluorophore, the probability for 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, fluorescence is observed in any appreciable amount in the focal volume, resulting in a high degree of rejection of out-of-focus objects. 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.
The use of infrared light to excite fluorophores in light-scattering tissue has added benefits. Longer wavelengths are scattered to a lesser degree than shorter ones, which is a benefit to high-resolution imaging. In addition, these lower-energy photons are less likely to cause damage outside of the focal volume. There are several caveats to using two-photon microscopy: Pulsed lasers are generally much more expensive, the microscope requires special optics to withstand the intense pulses, the two-photon absorption spectrum of a molecule may vary significantly from its one-photon counterpart, and wavelengths greater than 1400 nm may be significantly absorbed by the water in living tissue.