Practice question

Two-photon excitation uses pulsed infrared lasers where fluorescence arises only at the focal volume due to nonlinear absorption. Infrared light experiences reduced scattering and absorption by tissue, allowing photons to reach depths approaching one millimeter. Intrinsic optical sectioning eliminates need for pinhole and minimizes out-of-focus damage. This penetration depth far exceeds confocal, TIRF, or epifluorescence, which are limited to tens of microns. Consequently, neuroscientists employ two-photon for intravital imaging of cortical layers, tumor microenvironments, and developing embryos while maintaining high viability and subcellular resolution.