Effect of scattering on nonlinear optical scanning microscopy imaging of highly scattering media

The intensity of two-photon excited fluorescence (TPF) generated by ultrashort laser pulses was measured as a function of the depth of a focal point inside highly scattering media. The purpose was to investigate the spatial location of TPF in a scattering medium. Owing to the scattering, the intensity of the incident beam as well as the generated TPF signal was attenuated exponentially as the focal point was scanned into the medium. As the scattering strength of the medium was increased, the TPF was not confined to the focal region and had a wider distribution. These observations show that the scattering will result in the degradation of the ability of optical depth sectioning of nonlinear optical scanning microscopy.