Saturation modified point spread functions in two-photon microscopy

Excitation saturation can dramatically alter the effective imaging point spread function (PSF) in two‐photon fluorescence microscopy. The saturation‐modified PSF can have important implications for resolution in fluorescence imaging as saturation leads to both an increased fluorescence observation volume and an altered spatial profile for the PSF. We introduce here a computational approach to accurately quantify molecular excitation profiles that represent the modified imaging PSF in two‐photon microscopy under the influence of excitation saturation. An analytical model that accounts for pulsed laser excitation is developed to calculate the influence of saturation at any location within the excitation laser profile. The overall saturation modified molecular excitation profiles are then evaluated numerically. Our results demonstrate that saturation can play an important role in two‐photon fluorescence microscopy even with relatively modest excitation levels. Microsc. Res. Tech. 64:135–141, 2004. © 2004 Wiley‐Liss, Inc.