Out-of-focus fluorescence collection in two-photon microscopy of scattering media

Imaging depth in two-photon microscopy is ultimately limited by the out-of-focus fluorescence background which is collected indistinctly with the useful in-focus signal in scattering samples. We report on a complete Monte Carlo analysis of the collection process in two-photon imaging of turbid media. Epifluorescence collection efficiency of the microscope is shown to vary significantly from point to point inside the scattering medium, to the detriment of the in-focus signal and in favor to the background, lowering the signal-to-background ratio in the image. Moreover we found that this ratio is almost independent of the collecting path field of view for situations where the background overcomes the signal. Assuming that the out-of-focus background can be subtracted to the image, the signal-to-noise ratio in two-photon microscopy is forecast to benefit from enlarging the collection field of view, with a gain roughly proportional to this enlargement for deep imaging. Asymptotic behaviors of the Monte Carlo simulations are quantitatively interpreted from ballistic and diffusive approximations.