Premium
Saturation modified point spread functions in two‐photon microscopy
Author(s) -
Cianci Gianguido C.,
Wu Jianrong,
Berland Keith M.
Publication year - 2004
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20071
Subject(s) - saturation (graph theory) , excitation , two photon excitation microscopy , microscopy , point spread function , optics , fluorescence , fluorescence microscope , laser , fluorescence lifetime imaging microscopy , chemistry , materials science , molecular physics , physics , mathematics , quantum mechanics , combinatorics
Abstract 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.