Method to identify and minimize artifacts induced by fluorescent impurities in single-molecule localization microscopy
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Updated: Feb 3, 2026

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
Published on: April 25, 2025
Janel L Davis1, Biqin Dong1,2, Cheng Sun2
1Northwestern University, Department of Biomedical Engineering, Evanston, Illinois, United States.
Abstract:
The existence of fluorescent impurities has been a long-standing obstacle in single-molecule imaging, which results in sample misidentification and higher localization uncertainty. Spectroscopic single-molecule localization microscopy can record the full fluorescent spectrum of every stochastic single-molecule emission event. This capability allows us to quantify the spatial and spectral characteristics of fluorescent impurities introduced by sample preparation steps, based on which we developed a method to effectively separate fluorescent impurities from target molecules.
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