Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation.
Journal
Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604
Informations de publication
Date de publication:
14 Jun 2024
14 Jun 2024
Historique:
received:
29
03
2023
accepted:
25
04
2024
medline:
15
6
2024
pubmed:
15
6
2024
entrez:
14
6
2024
Statut:
aheadofprint
Résumé
Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.
Identifiants
pubmed: 38877317
doi: 10.1038/s41592-024-02293-8
pii: 10.1038/s41592-024-02293-8
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)
ID : R01GM098859
Informations de copyright
© 2024. The Author(s).
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