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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.

An effective Mie-scattering model is developed to deal with the scattering property of a spherical fractal aggregate consisting of scattering particles. In this model the scattered field of a scattering particle is given by the classical Mie-scattering theory. On the basis of the Monte Carlo simulation method, we determine the physical parameters of a scattering aggregate, the scattering efficiency Q, and the anisotropy value g, as well as their dependence on the size and the effective mean-free-path length of a scattering aggregate. Accordingly, photon migration through a microscope objective focused into a turbid medium including scattering aggregates is simulated to understand the effect of complex tissue on image quality.