Single-molecule microscopy for in-cell quantification of protein oligomeric stoichiometry

Protein organization modification plays a vital role in initiating signaling pathways, transcriptional regulation, and cell apoptosis regulation. Simultaneous quantification of oligomeric state and cellular parameters in the same cell, even though challenging, is required to understand their correlation at the molecular level. Recent advances of fluorescence protein and single-molecule localization microscopy enables the determination of localizations and oligomeric states of target proteins in cells. We reviewed the fluorescence intensity-based, localization-based, and photophysical property-based approaches for in-cell quantification of protein oligomeric stoichiometry. We discussed their working principles, applications, advantages, and limitations. These results also imply the combination of methodologies targeting different biological parameters at the single-cell level is essential to uncover the structure-function relationship at the molecular level.