Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy

Deep-tissue multiphoton microscopy (MPM) enables noninvasive optical imaging into the deep regions of the tissue in animal models in vivo. High-order MPM techniques, such as three- and four-photon fluorescence microscopy at the 1700-nm window, are emerging as promising imaging techniques for deeper penetration. Currently, signal depletion at large imaging depth sets the depth limit for these imaging techniques. As a result, how to further boost signal level is the key to achieving a larger imaging depth. Contrary to the previous thought that overfilling the back aperture of the objective yields the highest multiphoton fluorescence signal, in this paper, through numerical simulation, we show that, due to the effect of exponential decay of the excitation beam, the signal generation is maximized for certain underfilling of the back aperture of the objective lens. This will provide a simple strategy for signal generation maximization in deep-tissue MPM and potentially enables deeper imaging penetration into the tissue.