The optical scheme of a two photon microscope is not much different from a standard laser scanner confocal microscope. In both technologies a spotted light is produced by a laser source passing through a pinhole. Two galvanic mirrors then manipulate the angle of the light in order to scan the sample. The main differences concern the lack of the sample pinhole and the illumination source.
In confocal microscopy sample pinhole is in charge of filtering the fluorescence that originates at the focal plane. In two-photon microscopy this is not needed thanks to the different phenomenon used to excite fluorophores (Figure 3). The physical principle of two-photon absorption dictates that only at high photon density the phenomenon prevails over one photon absorption. The relation is a second order relation and implies that only when photons are concentrated enough fluorescence occurs (Figure 4). This brings to two photon microscopy a better resolution in the z axis when compared to confocal microscopes.
The second difference is related to the laser. While confocal microscopes laser emission is rather constant, two-photon microscopy uses pulsed laser in order to concentrate as much photons as possible in each single pulse.
The way the microscope illuminates the sample in a two-photon microscopy brings several advantages when compared to confocal microscopy. The main advantage is that two-photon microscopy can produce fluorescence with wavelengths (far-red, infrared) that are less subjected to light scattering. This significantly enhances the capability of the microscope to penetrate deeper in dense tissue. Longer wavelengths are also less toxic for cells and moreover the probability of secondary photochemical reactions are significantly much lower than what observed in confocal microscopy.