Disentangling Timescales of Molecular Kinetics with spFRET using ALEX-FCS.

ABSTRACT: Single-pair Förster resonance energy transfer (spFRET) probes the dynamics of molecular structures with (sub-)nanometer accuracy. When combined with fluorescence correlation spectroscopy (FCS), diffusion times and conformation lifetimes can be obtained. Alternating excitation (ALEX) further complements spFRET measurements on freely diffusing molecules, allowing for burst analysis, which can be used to reduce background signal without significant changes to the experimental setup. ALEX is particularly useful for extracting conformational dynamics, but extracting small differences in FRET levels and/or diffusion times can still be difficult for multi-species samples with fast or slow transition rates. Though the combination of spFRET, FCS and ALEX can help to constrain the fits of correlatio

SUBMITTER: Ernst J 

PROVIDER: S-EPMC12583438 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature