Spatial distribution of single-photon and two-photon fluorescence light in scattering media: Monte Carlo simulation

Three-dimensional fluorescence spatial distributions under single-photon and two-photon excitation within a turbid medium are studied with Monte Carlo simulation. It is demonstrated that two-photon excitation has an advantage of producing much less fluorescence light outside the focal region compared with single-photon excitation. With the increase of the concentration of scattering particles in a turbid medium, the position of the maximum fluorescence intensity point shifts from the geometric focal region toward the medium surface. Further studies show that the optical sectioning property of two-photon fluorescence microscopy is degraded in thick turbid media or when the numerical aperture of an objective becomes low.