Spectral cross-cumulants for multicolor super-resolved SOFI imaging.

Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution and the sampling can be increased up to n-fold in all spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increases the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and experiments in fixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property. Here, the authors generalize cumulant analysis by extending it into the spectral domain to allow multicolour super-resolution optical fluctuation imaging. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing allows denser spectral and spatial sampling of the super-resolved image.