Monte Carlo Simulation of Image Depth Improvement by Two-color Two-photon Fluorescence Microscopy

Abstract

Out of focus fluorescence is a dominant limiting factor in the improvement of imaging depth in two-photon fluorescence microscopy. Here we propose a new two-color two-photon fluorescence microscopy scheme, in which two trains of laser pulses with different wavelengths are concentric and complementary. These two beams are overlapped both in space and time only in the focal area. Monte Carlo simulation demonstrates that this scheme can effectively reject out-of-focus fluorescence excitation, and thus has great potential for providing significant increment of imaging-depth.