Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores
| Autor: | Thiemo Spielmann, Andriy Chmyrov, Heike Hevekerl, Jerker Widengren |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Fluorophore
Biophysics Fluorescence correlation spectroscopy 02 engineering and technology 010402 general chemistry Photochemistry 01 natural sciences 7. Clean energy Molecular physics chemistry.chemical_compound Fluorescence Resonance Energy Transfer Materials Chemistry Physical and Theoretical Chemistry Triplet state Fluorescent Dyes Range (particle radiation) Chemistry Potassium Iodide 021001 nanoscience & nanotechnology Acceptor Fluorescence Biofysik 0104 chemical sciences Surfaces Coatings and Films Kinetics Förster resonance energy transfer Quantum Theory 0210 nano-technology Algorithms Excitation |
| Zdroj: | The Journal of Physical Chemistry B The Journal of Physical Chemistry B; Vol 115 |
| ISSN: | 1520-5207 1520-6106 |
| DOI: | 10.1021/jp206770s |
| Popis: | Inter- or intramolecular distances of biomolecules can be studied by Forster 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. QC 20150624 |
| Databáze: | OpenAIRE |
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