ArticleBiophysical journal2010
Optimizing methods to recover absolute FRET efficiency from immobilized single molecules.
Article in Biophysical journal, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 69 papers.
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Who cites it
69 citing papers in PubMed, 117 citations in OpenAlex.
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- Multiplexed single-molecule characterization at the library scale.Nature protocols · 2026Review
- Correlating membrane-protein dynamics with function: Integrating bioinformatics, molecular dynamics, and single-molecule FRET.Protein science : a publication of the Protein Society · 2025Article
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- SNARE disassembly requires Sec18/NSF side-loading.bioRxiv : the preprint server for biology · 2025Article
- Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology.Journal of microscopy · 2025Review
- Direct Quantification of Protein-Protein Interactions in Living Bacterial Cells.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Article
- Understanding the Mechanism of Bent DNA Amplifying Sensors Using All-Atom Molecular Dynamics Simulations.Biosensors · 2025Article
- Structural Dynamics of SNARE Complex Assembly in the Ribbon Synapses Observed by smFRET.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Article
- Developing Multichannel smFRET Approach to Dissecting Ribosomal Mechanisms.Chemical & biomedical imaging · 2024Article
- Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation.Nature methods · 2024Article
- Full-length single-molecule protein fingerprinting.Nature nanotechnology · 2024Article
- Reply to: On the statistical foundation of a recent single molecule FRET benchmark.Nature communications · 2024Article
- Measurement of Forces Acting on Single T-Cell Receptors.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Frustration Between Preferred States of Complementary Trinucleotide Repeat DNA Hairpins Anticorrelates with Expansion Disease Propensity.Journal of molecular biology · 2023Article
- Small GTPase Cdc42, WASP, and scaffold proteins for higher-order assembly of the F-BAR domain protein.Science advances · 2023Article
- Single-photon smFRET: II. Application to continuous illumination.Biophysical reports · 2023Article
9 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected by differences in fluorophore quantum yield. Recovering the absolute FRET efficiency requires normalization for these effects to account for differences between the donor and acceptor fluorophores in their quantum yield and detection efficiency. Without this correction, FRET is consistent across multiple experiments only if the photophysical and instrument properties remain unchanged. Here we present what is, to our knowledge, the first systematic study of methods to recover the true FRET efficiency using DNA rulers with known fluorophore separations. We varied optical elements to purposefully alter observed FRET and examined protein samples to achieve quantum yields distinct from those in the DNA samples. Correction for calculated instrument transmission reduced FRET deviations, which can facilitate comparison of results from different instruments. Empirical normalization was more effective but required significant effort. Normalization based on single-molecule photobleaching was the most effective depending on how it is applied. Surprisingly, per-molecule gamma-normalization reduced the peak width in the DNA FRET distribution because anomalous gamma-values correspond to FRET outliers. Thus, molecule-to-molecule variation in gamma has an unrecognized effect on the FRET distribution that must be considered to extract information on sample dynamics from the distribution width.
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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.