1 research outputs found
Stuck Photoswitching Event Analysis and Correction for Superresolution Single-Molecule Localization Microscopy
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 10β20 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