Improving single-molecule FRET measurements by confining molecules in nanopipettes

J. Vogelsang; S. Doose; M. Sauer; P. Tinnefeld

doi:10.1117/12.727842

13 July 2007 Improving single-molecule FRET measurements by confining molecules in nanopipettes

J. Vogelsang, S. Doose, M. Sauer, P. Tinnefeld

Proceedings Volume 6633, Biophotonics 2007: Optics in Life Science; 66331L (2007) https://doi.org/10.1117/12.727842
Event: European Conferences on Biomedical Optics, 2007, Munich, Germany

Abstract

In recent years Fluorescence Resonance Energy Transfer (FRET) has been widely used to determine distances, observe distance dynamics, and monitor molecular binding at the single-molecule level. A basic constraint of single-molecule FRET studies is the limited distance resolution owing to low photon statistics. We demonstrate that by confining molecules in nanopipettes (50-100 nm diameter) smFRET can be measured with improved photon statistics reducing the width of FRET proximity ratio distributions (PRD). This increase in distance resolution makes it possible to reveal subpopulations and dynamics in biomolecular complexes. Our data indicate that the width of PRD is not only determined by photon statistics (shot noise) and distance distributions between the chromophores but that photoinduced dark states of the acceptor also contribute to the PRD width. Furthermore, acceptor dark states such as triplet states influence the accuracy of determined mean FRET values. In this context, we present a strategy for the correction of the shift of the mean PR that is related to triplet induced blinking of the acceptor using reference FCS measurements.

Citation Download Citation

J. Vogelsang, S. Doose, M. Sauer, and P. Tinnefeld "Improving single-molecule FRET measurements by confining molecules in nanopipettes", Proc. SPIE 6633, Biophotonics 2007: Optics in Life Science, 66331L (13 July 2007); https://doi.org/10.1117/12.727842

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