Fluorescence saturation imaging microscopy: molecular fingerprinting in living cells using two-photon absorption cross section as a contrast mechanism.

Imaging of molecular-specific photophysical parameters such as fluorescence intensity, emission band shape, or fluorescence decay is widely used in biophysics. Here we propose a method for quantitative mapping of another molecular-specific parameter in living cells, two-photon absorption cross section, based on the fluorescence saturation effect. Using model dye solutions and cell culture, we show that the analysis of the fluorescence signal dependencies on the intensity of two-photon excitation within the range typical for routine two-photon microscopy experiments allows one to reconstruct two-photon absorption cross section maps across the sample. We believe that the absorption cross section contrast visualized by the proposed fluorescence saturation imaging microscopy could be a new tool for studying processes in living cells and tissues.