Two Photon Microscopy
Two photon microscopy is a fluorescence imaging technique that can be employed to increase the imaging depth and can be beneficial for three dimensional imaging. Two-photon microscopy uses infrared light that minimises light absorption and scattering in the tissue thereby increasing imaging depth and can in these cases be superior to confocal microscopy.
The concept of two-photon microscopy is well established and is built around the concept that in order for a fluorophore to fluoresce, it must receive a quantum (or packet) of energy. This is essentially photons of a particular wavelength (and therefore specific energy), only these photons are capable of exciting electrons into the required higher energy state needed to induce fluorescence (see description of fluorescence). The two-photon idea proposes that if two photons of around half the wavelength (and therefore energy) are simultaneously absorbed by the fluorophore there should be enough energy to excite electrons into the higher energy level and therefore induce fluorescence (figure 1).
However, the probability of near simultaneous absorption of the required two photons is very low, to get around this, a high photon flux is required and is provided by the output of a focused pulsed (femtosecond) tuneable infrared laser. An added benefit of this approach is that excitation of fluorophores outside of the focal plane is minimised primarily because of the reduced chance of simultaneous multiple photon absorption by the fluorophore.
Two-photon microscopy is an ideal solution for three-dimensional image capture and unlike confocal microscopy does not require a detector pinhole. In addition to this, because excitation is typically in the infrared region of the spectrum, absorption by cells / tissues is minimal thus, in theory two-photo microscopy is a good live-cell imaging technique. Having said that, two-photon microscopy may not work well with all fluorophores and the two-photon absorption spectrum of the fluorophore may be different to that when excited in single-photon mode. For thin specimens, two-photon microscopy may not provide any added benefit over conventional confocal microscopy using shorter excitation (single –photon) wavelengths.
The Leica SP2 UV (upright) confocal microscope is equipped for two photon imaging in addition of combination with conventional confocal imaging.
