Super-resolution fluorescence imaging techniques allow optical imaging of specimens beyond the diffraction limit of light. Super-resolution optical fluctuation imaging (SOFI) relies on computational analysis of stochastic blinking events to obtain a super-resolved image. As with some other super-resolution methods, this strong dependency on computational analysis can make it difficult to gauge how well the resulting images reflect the underlying sample structure. We herein report SOFIevaluator, an unbiased and parameter-free algorithm for calculating a set of metrics that describes the quality of super-resolution fluorescence imaging data for SOFI. We additionally demonstrate how SOFIevaluator can be used to identify fluorescent proteins that perform well for SOFI imaging under different imaging conditions.

SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data