A Pairwise Distance Distribution Correction (DDC) algorithm for blinking-free super-resolution microscopy
2019
Abstract In single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence. Intermittent emissions create multiple localizations belonging to the same molecule, a phenomenon known as blinking. Blinking distorts SMLM images and confound quantitative interpretations by forming artificial nanoclusters, which are often interpreted as true biological assemblies. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate fluorophore blinking in superresolution imaging without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequence by using the distances between localizations separated by a time much longer than the average fluorescence survival time. We show that using the true pairwise distribution we can define and then maximize the likelihood of obtaining a particular set of localizations without blinking and generate an accurate reconstruction of the true underlying cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous existing blinking correction methodologies, resulting in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters. The simplicity and robustness of DDC will enable its wide application in SMLM imaging, providing the most accurate reconstruction and quantification of SMLM images to date.
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