ArticleBiophysical journal2020
Correction of Systematic Bias in Single Molecule Photobleaching Measurements.
Article in Biophysical journal, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed, 11 citations in OpenAlex.
- Determining the Stoichiometry of Amyloid Oligomers by Single-Molecule Photobleaching.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Single-molecule microscopy for in-cell quantification of protein oligomeric stoichiometry.Current opinion in structural biology · 2021Review
- Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches.Computational and structural biotechnology journal · 2021Review
- Single Molecule Measurements of the Accessibility of Molecular Surfaces.Frontiers in molecular biosciences · 2021Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Single molecule photobleaching is a powerful technique to measure the number of fluorescent units in subresolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photobleaching can occur before the sample is appropriately placed and focused. Such "prebleaching" can introduce a strong systematic bias toward smaller oligomers. Quantitative correction of prebleaching is known to be an ill-posed problem, limiting the utility of the technique. Here, we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the prebleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human islet amyloid polypeptide. Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B > 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.
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Registered trials
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