Role of scattering and Nonlinear Effects in the illumination and the photobleaching distribution profiles

Nonlinear optical scanning microscopy has become a useful tool for living tissue imaging. Biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. While performing imaging of biological scattering tissues in nonlinear excitation regime, the localization of the maximum two-photon excitation (2PE) intensity was found to shift closer to the surface and the 2PE imaging depth limit appears strongly limited by near-surface fluorescence. In this work, we computed the illumination and the photobleaching distribution to characterize the effects induced by scattering. An experimental test has been carried out by imaging, with 0.9 NA objective, thick scattering fluorescent immobile sample (polyelectrolyte gel) as a phantom for biological samples. Results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface.