Two-photon excitation (TPE) microscopy enables imaging of living tissues up to a depth of one millimeter, which is 6 to 10-fold deeper than with confocal microscopy. Like confocal microscopy, TPE microscopy is capable of optical sectioning, allowing three-dimensional reconstruction of a specimen.

Principle

Similar to confocal microscopy, TPE microscopy is a laser-scanning imaging technique in which excitation light is focused into a very small spot and is scanned across the specimen to reconstitute a 2D image. Both techniques excel in reducing out-of-focus background fluorescence, which increases the signal-to-noise ratio. In confocal microscopy, a pinhole is placed in front of the detector to exclude fluorescence emitted above and below the focal plane. However, this strategy reaches its limit when one wants to image deeper than 50 to 100 µm because most of the excitation light is absorbed or scattered by the tissue before it reaches the focal plane. To image at these depths, a greater laser output power would be necessary. However, it would cause significant photobleaching and phototoxicity issues, particularly in live specimens, and the increased fluorescence due to excitation scattering will lead to a background haze that reduces contrast.

TPE microscopy is based on the ability of fluorophores to absorb two photons at a wavelength about twice that required for single-photon excitation in confocal microscopy.

Because these events must happen nearly simultaneously, they are very rare and only occur with extremely high photon density, thereby restricting two-photon excitation at the focal plane (see figure below). It is because fluorophores are only excited in the focal plane that out-of-focus absorption is not an issue in TPE microscopy. As a result, the signal-to-noise ratio is increased and photobleaching/photodamage is minimal. In addition, the ability of longer wavelengths of light to penetrate deeper into tissues (=less excitation scattering) enables deeper imaging performances.

Advantages

• Deep tissue imaging
• Less photodamage
• The red light utilized in 2-photon excitation is less detrimental for living tissues than the UV light used to image UV fluorophores (e.g. NAD(P)H) or to photoactivate caged compounds (e.g. caged glutamate or calcium)

Limitations

• The maximal spatial resolution achievable is reduced compared to conventional microscopy because of the longer wavelengths used and the lower numerical aperture of 2-photon objectives.