Two-photon excitation microscopy

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Two-photon excitation microscopy is a technique that allows imaging living tissue up to a depth of one millimeter. 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 Göppert-Mayer (b. 1906) in her doctoral dissertation (1931). 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 fluorescence. The probability of the simultaneous absorption of two photons is extremely low. Therefore a high flux of excitation photons is typically required.

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 ~ 100 femtoseconds and a repetition rate of ~ 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.

A diagram of a two-photon microscope

The most commonly used fluorophores have excitation spectra in the 400-500 nm range, whereas the laser used to excite the fluorophores lies in the ~700-1000 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 spectra). Because two photons need to be absorped to excite a fluorophore, the probability of emission is related to the intensity squared of the excitation beam. Consequently, the excited fluorophores are mostly confined to the focal volume. The use of infrared light to excite fluorophores in light-scattering tissue has added benefits. Longer wavelengths are scattered less, 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.