Scattering effect on the imaging depth limitin two-photon fluorescence microscopy

A model for the propagation of a focused light beam in a strongly scattering medium is used to analyse various factors that limit the capability of two-photon fluorescence microscopy (TPFM) to image deep sections of optically thick biological specimens. The TPFM imaging depth is shown to be limited by three main factors: (1) beam broadening as a result of multiple small-angle scattering, leading to a loss of submicron lateral resolution; (2) strong near-surface fluorescence at large imaging depths as a result of the increase in average source power in order to compensate for scattering losses; (3) reduction in useful two-photon fluorescence signal level because of the exponential attenuation of the excitation power. The influence of these factors is examined in a small-angle diffusion approximation of radiative transfer theory. The first two of them are shown to set a fundamental TPFM limit, whereas the last is an instrumental limitation and appears to be the most critical to state-of-the-art commercial two-photon laser scanning microscopy systems for the vast majority of fluorophores in current use.