Penetration depth of single-, two-, and three-photon fluorescence microscopic imaging through human cortex structures: Monte Carlo simulation.

Penetration depth is investigated in terms of the performance of transverse image resolution and signal level in human cortex under single-, two-, and three-photon fluorescence microscopy. Simulation results show that, in a double-layer human cortex structure consisting of gray and white matter media, the signal level is strongly affected by the existence of the white matter medium under three-photon excitation. Compared with three-photon excitation, two-photon excitation keeps a better signal level and sacrifices a slight degradation in image resolution. In a thick gray matter medium, a penetration depth of 1500 microm with a near-diffraction-limited resolution is obtainable under three-photon excitation. It is also demonstrated that the numerical aperture has a slight influence on image resolution and signal level under two- and three-photon excitation because of the nonlinear nature in the excitation process.