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

Abstract

Imaging of molecular-specific photophysical parameterssuch as fluorescence intensity, emission band shape, or fluorescence decay is widely used in biophysics. Here we proposea method for quantitative mapping of another molecularspecific parameter in living cells, two-photon absorptioncross section, based on the fluorescence saturation effect.Using model dye solutions and cell culture, we show thatthe analysis of the fluorescence signal dependencies on theintensity of two-photon excitation within the range typicalfor routine two-photon microscopy experiments allows one toreconstruct two-photon absorption cross section maps acrossthe sample. We believe that the absorption cross sectioncontrast visualized by the proposed fluorescence saturationimaging microscopy could be a new tool for studying processes in living cells and tissues. © 2022 Optica PublishingGroup