Limiting Factors on Image Quality in Imaging through Turbid Media under Single-photon and Two-photon Excitation.

The effect of multiple scattering in a turbid medium on single-photon and two-photon fluorescence microscopy is experimentally investigated for different scattering characteristics including scattering anisotrophy and optical thickness of a turbid medium. It is demonstrated that two-photon excitation can provide significant improvement in penetration depth through turbid media, due to reduced scattering experienced by the excitation beam. It is also shown that the limiting factor in obtaining high-quality images under single-photon excitation is the fast degradation of image resolution caused by multiple scattering, while for two-photon excitation it is limited by the degradation of image contrast due to the reduction in fluorescence strength.