Two-photon microscopy with enhanced contrast and resolution.

A method combining the saturation effect with the ratio concerned quadratic intensity weighted subtraction (RQIWS) algorithm for resolution and contrast enhancement in a two-photon microscopy system is presented in this paper. In the proposed method, the saturation effect is utilized to get a profile-extended solid spot and a center-shrunken doughnut-shaped spot for a smaller effective point spread function, which enhances the resolution of the system. The RQIWS algorithm uses the intensity ratio of the two original images acquired respectively with the solid spot and the doughnut-shaped spot as one of the subtraction parameters and takes the fluorescence quadratic dependence of excitation intensity into account for better subtraction results compared with the intensity weighted subtraction algorithm in a two-photon excitation system. The capability in the enhancement of resolution and contrast of the method proposed is demonstrated successfully both by theoretical simulations and experimental results.