title: Förster resonance energy transfer beyond 10 nm: exploiting the triplet state kinetics of organic fluorophores.
abstract: Inter- or intramolecular distances of biomolecules can be studied by Förster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm, and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleed-through, thereby tolerating a high excess (up to 100-fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50% in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores, and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in vitro and in vivo FRET-based spectroscopy and imaging.
authors.affiliation: Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Heike
authors.initials: H
authors.lastname: Hevekerl
authors.forename: Thiemo
authors.initials: T
authors.lastname: Spielmann
authors.forename: Andriy
authors.initials: A
authors.lastname: Chmyrov
authors.forename: Jerker
authors.initials: J
authors.lastname: Widengren
mesh_terms: D000465:Algorithms; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D011193:Potassium Iodide / Q000737:chemistry; D011789:Quantum Theory
chemical_list: D005456:Fluorescent Dyes; D011193:Potassium Iodide