ArticleBiophysical journal2015
Automated Analysis of Single-Molecule Photobleaching Data by Statistical Modeling of Spot Populations.
Article in Biophysical journal, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- Single-Molecule Stoichiometry of Supramolecular Complexes.Journal of the American Chemical Society · 2024Article
- Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy.Analytical chemistry · 2023Article
- Modeling Non-additive Effects in Neighboring Chemically Identical Fluorophores.The journal of physical chemistry. B · 2022Article
- Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK.The Journal of cell biology · 2022Article
- Diffraction-Limited Molecular Cluster Quantification with Bayesian Nonparametrics.Nature computational science · 2022Article
- Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer.Cancer discovery · 2022Article
- Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3.The EMBO journal · 2022Article
- Photobleaching step analysis for robust determination of protein complex stoichiometries.Molecular biology of the cell · 2021Article
- Smart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames.Nucleic acids research · 2021Article
- Tetrameric architecture of an active phenol-bound form of the AAANature communications · 2020Article
- Determining the correct stoichiometry of Kv2.1/Kv6.4 heterotetramers, functional in multiple stoichiometrical configurations.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Quantitative microscopy based on single-molecule fluorescence.Current opinion in chemical biology · 2017Review
- Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis.Chemical reviews · 2017Article
- Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching.The Journal of biological chemistry · 2016Article
- A novel method to accurately locate and count large numbers of steps by photobleaching.Molecular biology of the cell · 2016Article
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Authors and funding
8 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The number of fluorophores within a molecule complex can be revealed by single-molecule photobleaching imaging. A widely applied strategy to analyze intensity traces over time is the quantification of photobleaching step counts. However, several factors can limit and bias the detection of photobleaching steps, including noise, high numbers of fluorophores, and the possibility that several photobleaching events occur almost simultaneously. In this study, we propose a new approach, to our knowledge, to determine the fluorophore number that correlates the intensity decay of a population of molecule complexes with the decay of the number of visible complexes. We validated our approach using single and fourfold Atto-labeled DNA strands. As an example we estimated the subunit stoichiometry of soluble CD95L using GFP fusion proteins. To assess the precision of our method we performed in silico experiments showing that the estimates are not biased for experimentally observed intensity fluctuations and that the relative precision remains constant with increasing number of fluorophores. In case of fractional fluorescent labeling, our simulations predicted that the fluorophore number estimate corresponds to the product of the true fluorophore number with the labeling fraction. Our method, denoted by spot number and intensity correlation (SONIC), is fully automated, robust to noise, and does not require the counting of photobleaching events.
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