Stuck Photoswitching Event Analysis and Correction for Superresolution Single-Molecule Localization Microscopy

Abstract

Numerous emerging derivatives of superresolution techniques have been proposed to conduct nanoscopic analysis for studying cellular structure. Single-molecule localization microscopy (SMLM) can routinely achieve a superior spatial resolution of 10−20 nm, enabling the observation of protein localization with molecular details. However, the stochastic detection of fluorophores often causes image artifacts from the spurious localizing process due to the disproportionate counting of single-molecule events to molecule numbers. The overaccumulated localizations, which lead to excessively high intensities in rendered images, could hinder the visualization of actual molecular distribution; therefore, the image artifacts stemming from uneven photoswitching events remain unsolved. Here, we propose a simple approach to address this general issue in SMLM with the overaccumulation of localizations from fluorophores at the prolonged emissive state. Our strategy involves extracting the photoswitching pattern from individual single-molecule events. Subsequently, we remove signals from the long-lived emissive state of fluorophores by applying optimized linking and cutoff lengths, thereby correcting localization artifacts. To demonstrate the practicality of our proposed method, we adopted this approach to restore the superresolution results of various cellular structures and organelles. Notably, the treated images manifest superresolved details and balanced intensity; this allows precise interpretation of molecular clusters and suggests its role in imaging processing of SMLM.