  Super-resolution optical fluctuation imaging (SOFI) 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 as well as the sampling can be increased up to n-fold in
all three spatial dimensions. In this study, we extend the cumulant analysis
into the spectral domain and propose a novel multicolor super-resolution
scheme. The simultaneous acquisition of two spectral channels followed by
spectral cross-cumulant analysis and unmixing increase 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 multiple
experiments with different cellular structures, fluorophores and filter sets.
Based on an eigenvalue/ vector analysis we propose a scheme for an optimized
spectral filter choice. Overall, our methodology provides a novel route for
easy-to-implement multicolor sub-diffraction imaging using standard microscopes
while conserving the spatial super-resolution property. This makes simultaneous
multiplexed super-resolution fluorescence imaging widely accessible to the life
science community interested to probe colocalization between two or more
molecular species.
