Multiphoton microscopy (MPM) encompasses a group of microscopy modalities that are characterized by a nonlinear optical process where two or three photons are utilized for quasi-simultaneous absorption with emission of a single photon (Ustione and Piston, 2011). In contrast to single photon excitation, such as CFM, where the relationship between the emission and excitation amplitude is linear, MPM relies on nonlinear interaction (second or third order relation from excitation to emission) between photons and the molecule (Masters, 2006), resulting in several advantages and important features for cellular imaging. The longer wavelength of infrared light employed by MPM for excitation leads to higher penetration depth into the tissue sample as compared to CM because of the reduced diffraction of light. Generally, the imaging depth of MPM is ~400 pm, though it can reach up to ~1 mm and even higher depending on the transparency of the sample. The multiphoton excitation requires a high photon density from the illumination beam, which is only achieved at the focal volume, thus producing a natural pinhole effect that eliminates the out-of-focus light. Since a spatial pinhole aperture in CM rejects part of the light from the focal region inside the sample due to scattering, in comparison, MPM collects all emission light from the multiphoton excitation, minimizing the signal loss. Also, compared with CFM, where molecules along the whole illumination path are excited, MPM possesses significantly less photo-bleaching effect due to excitation only in the focal zone (Niesner and Hauser, 2011).
Because the wavelength of illumination is longer than the one used for CFM, the focal size that can be achieved in MPM is relatively larger, resulting in lower spatial resolution, but still featuring at the micron and submicron level (Scientist and Technology, 2008). Specifically, for two-photon excitation, the spatial resolution is about twice of CFM. As the pinhole effect in MPM is intrinsically achieved by focusing the light, the spatial resolution of MPM is mainly determined by the numerical aperture of the objective lens. Similar to CM, the depthwise sectioning of MPM is obtained with axial movement of the imaging lens, and laser scanning is generally employed for a 3-D reconstruction of the sample. Multiphoton excitation is a powerful approach that has served as the basis for a number of imaging modalities (Masters, 2006). Here, we focus our discussions on two-photon fluorescence and second harmonic generation microscopy, which have both been used largely in tissue engineering.