Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium

Image contrast enhancement is investigated for two-photon excitation fluorescence images of a microscopic sample that is buried underneath a turbid medium. The image contrast, which deteriorates rapidly with sample depth because of scattering loss, is enhanced by an increase in the average excitation power of the focused Gaussian (the TEM,, mode) beam according to a compensation relation that has been derived by use of a Monte Carlo analysis of the scattering problem. A correct increase in the excitation power results in a detected fluorescence signal that remains invariant with sample depth. The scheme is demonstrated on images of DAPI-stained nuclei cells viewed underneath a suspension of 0.105-mu m-diameter polystyrene spheres. (C) 1998 Optical Society of America.