Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation.

ABSTRACT: 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

SUBMITTER: Pati AK 

PROVIDER: S-EPMC11239528 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature