Minimizing Molecular Misidentification in Imaging Low-Abundance Protein Interactions Using Spectroscopic Single-Molecule Localization Microscopy.
Anal Chem
; 94(40): 13834-13841, 2022 10 11.
Article
in En
| MEDLINE
| ID: mdl-36165784
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biological Phenomena
/
Single Molecule Imaging
Language:
En
Journal:
Anal Chem
Year:
2022
Document type:
Article
Affiliation country:
United States
Country of publication:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
/
US
/
USA