2022 journal article

Minimizing Molecular Misidentification in Imaging Low-Abundance Protein Interactions Using Spectroscopic Single-Molecule Localization Microscopy

Analytical Chemistry.

co-author countries: China 🇨🇳 United States of America 🇺🇸
MeSH headings : Biological Phenomena; Fluorescent Dyes / chemistry; Microscopy, Fluorescence / methods; Single Molecule Imaging / methods; Staurosporine / pharmacology
Source: ORCID
Added: March 13, 2023

Super-resolution microscopy can capture spatiotemporal organizations of protein interactions with resolution down to 10 nm; however, the analyses of more than two proteins involving low-abundance protein are challenging because spectral crosstalk and heterogeneities of individual fluorescent labels result in molecular misidentification. Here we developed a deep learning-based imaging analysis method for spectroscopic single-molecule localization microscopy to minimize molecular misidentification in three-color super-resolution imaging. We characterized the 3-fold reduction of molecular misidentification in the new imaging method using pure samples of different photoswitchable fluorophores and visualized three distinct subcellular proteins in U2-OS cell lines. We further validated the protein counts and interactions of TOMM20, DRP1, and SUMO1 in a well-studied biological process, Staurosporine-induced apoptosis, by comparing the imaging results with Western-blot analyses of different subcellular portions.