Factors affecting ultimate imaging depth of two-photon fluorescence microscopy in scattering medium

Different aspects of multiple small-angle scattering effect on two-phonon fluorescence microscopy (2PFM) imaging ability are discussed in this paper. We focus on theoretical evaluation of the maximum accessible imaging depth. There are three main factors which potentially restrict imaging depth: i) decay of tightly focused excitation beam caused by scattering and accompanied by loss of diffraction-limited resolution; ii) out-of focus fluorescence originated from excessive illumination of the sample surface which is required to compensate for the lack of peak intensity inside scattering medium; iii) decrease of signal-to noise ratio of fluorescence signal due to Beer-Bouguer-Lambert law decrease of excitation intensity. Based on small-angle diffusive approximation of radiation transfer theory we compared the influence of these factors and found out that the first two factors define fundamental limitation of 2PEM potentialities in scattering medium while the last one provides principal instrumental limitation which prevails in state-of the-art commercial laser scanning microscopy systems.