ArticleMolecular biology of the cell2021
Photobleaching step analysis for robust determination of protein complex stoichiometries.
Article in Molecular biology of the cell, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed, 43 citations in OpenAlex.
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- Mutual antagonism between PRC1 condensates and SWI/SNF in chromatin regulation.Molecular cell · 2026Article
- MERTK coordinates efferocytosis by regulating integrin localization and activation.Journal of cell science · 2026Article
- Toll-like receptor signaling outcome is determined by the stoichiometry of the endogenous TRIFosome.Science advances · 2026Article
- A Pseudomonas aeruginosa quorum-sensing inducer controls lung permeability in establishing chronic infection via EGFR.Nature communications · 2025Article
- Quantifying Intracellular Distributions of HaloTag-Labeled Proteins With SDS-PAGE and Epifluorescence Microscopy.Bio-protocol · 2025Article
- Photoactivation of BODIPY Fluorescence with Green Light.The Journal of organic chemistry · 2025Article
- Bayesian Inference of Binding Kinetics from Fluorescence Time Series.The journal of physical chemistry. B · 2025Article
- A Bayesian Model to Count the Number of Two-State Emitters in a Diffraction Limited Spot.Nano letters · 2025Article
- Therapeutic application of circular RNA aptamers in a mouse model of psoriasis.Nature biotechnology · 2025Article
- Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin.eLife · 2024Article
- ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting.Nature methods · 2024Article
- Advanced multiparametric image spectroscopy and super-resolution microscopy reveal a minimal model of CD95 signal initiation.Science advances · 2024Article
- Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin.bioRxiv : the preprint server for biology · 2024Article
- Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.Journal of medicinal chemistry · 2024Article
- CaCommunications biology · 2024Article
- Modularity of PRC1 composition and chromatin interaction define condensate properties.Molecular cell · 2024Article
Corrections and comments
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Authors and funding
7 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has been significantly improved with more-sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high-quality data. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labeling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the developed methods with experimental data acquired on DNA origami labeled with defined fluorophore numbers, demonstrating counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis by counting labeled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated manner.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.