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Single-Molecule FRET: Principles and Analysis

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Fluorescence Spectroscopy and Microscopy in Biology

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 20))

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Abstract

Förster resonance energy transfer (FRET) is a powerful spectroscopic technique to study conformational changes of nucleic acids and proteins and their molecular interactions. When combined with a single-molecule approach, FRET has the distinct advantage that it can monitor the conformational heterogeneity and dynamics of individual molecules and enable the observation of short-lived molecular intermediates usually hidden in ensemble experiments. This in turn makes single-molecule FRET an interesting tool for dynamic structural biology.

This chapter presents the principles of single-molecule FRET spectroscopy and the added information it gives compared to ensemble FRET spectroscopy. We describe different experimental implementations, primarily focusing on intensity-based approaches. Fluorescence from single molecules requires careful experimental procedures to maximize the inherently low signal intensity, and meticulous data analysis, which is introduced in this chapter, to quantify FRET detection. We comment on advantages and limitations of the technique, and its strength is illustrated by two application examples.

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Acknowledgments

We thank the reviewer for his/her feedback. We would like to acknowledge support from the Novo Nordisk Foundation (grant NNF20OC0061417) and from the Danish Council for Independent Research (grant DFF-9040-00323B).

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  1. Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark

    B. Israels, L. M. Lund & V. Birkedal

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  1. B. Israels
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  2. L. M. Lund
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  1. J. HeyrovskĂ˝ Institute of Physical Chemistry, Prague, Czech Republic

    Radek Ĺ achl

  2. J. HeyrovskĂ˝ Institute of Physical Chemistry, Prague, Czech Republic

    Mariana Amaro

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Israels, B., Lund, L.M., Birkedal, V. (2022). Single-Molecule FRET: Principles and Analysis. In: Ĺ achl, R., Amaro, M. (eds) Fluorescence Spectroscopy and Microscopy in Biology. Springer Series on Fluorescence, vol 20. Springer, Cham. https://doi.org/10.1007/4243_2022_32

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