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

Yang Zhang, Gaoxiang Wang, Peizhou Huang, Edison Sun, Junghun Kweon, Qianru Li, Ji Zhe, Leslie L. Ying, Hao F. Zhang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)13834-13841
Number of pages8
JournalAnalytical Chemistry
Volume94
Issue number40
DOIs
StatePublished - Oct 11 2022

ASJC Scopus subject areas

  • Analytical Chemistry

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