A pairwise distance distribution correction (DDC) algorithm to eliminate blinking-caused artifacts in SMLM

• DOI: 10.1038/s41592-021-01154-y
• Publication Date: 2021-05-31T21:11:59Z
• Authors: Christopher H. Bo...; Xinxing Yang; Shreyasi Thakur; Xiaoli Weng; Brian Tenner; Ryan McQuillen; Brian Ross; Matthew Wooten; Xin Chen; Jin Zhang; Elijah Roberts; Melike Lakadamyali; Jie Xiao
• Keywords: Stochastic Processes; Microscopy, Fluorescence; 0206 medical engineering; 02 engineering and technology; Artifacts; Algorithms; Single Molecule Imaging; Fluorescent Dyes

Single-molecule localization microscopy (SMLM) relies on the blinking behavior of a fluorophore, which is the stochastic switching between fluorescent and dark states. Blinking creates multiple localizations belonging to the same fluorophore, confounding quantitative analyses and interpretations. Here we present a method, termed distance distribution correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on obtaining the true pairwise distance distribution of different fluorophores naturally from the imaging sequence by using 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 maximize the likelihood, obtaining a set of localizations void of blinking artifacts. DDC results in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications, enabling accurate reconstruction and quantification of SMLM images.