Issue 35, 2014

Fast single-molecule FRET spectroscopy: theory and experiment

Abstract

Single-molecule spectroscopy is widely used to study macromolecular dynamics. Although this technique provides unique information that cannot be obtained at the ensemble level, the possibility of studying fast molecular dynamics is limited by the number of photons detected per unit time (photon count rate), which is proportional to the illumination intensity. However, simply increasing the illumination intensity often does not help because of various photophysical and photochemical problems. In this Perspective, we show how to improve the dynamic range of single-molecule fluorescence spectroscopy at a given photon count rate by considering each and every photon and using a maximum likelihood method. For a photon trajectory with recorded photon colors and inter-photon times, the parameters of a model describing molecular dynamics are obtained by maximizing the appropriate likelihood function. We discuss various likelihood functions, their applicability, and the accuracy of the extracted parameters. The maximum likelihood method has been applied to analyze the experiments on fast two-state protein folding and to measure transition path times. Utilizing other information such as fluorescence lifetimes is discussed in the framework of two-dimensional FRET efficiency-lifetime histograms.

Graphical abstract: Fast single-molecule FRET spectroscopy: theory and experiment

Supplementary files

Article information

Article type
Perspective
Submitted
05 Jun 2014
Accepted
24 Jul 2014
First published
04 Aug 2014

Phys. Chem. Chem. Phys., 2014,16, 18644-18657

Fast single-molecule FRET spectroscopy: theory and experiment

H. S. Chung and I. V. Gopich, Phys. Chem. Chem. Phys., 2014, 16, 18644 DOI: 10.1039/C4CP02489C

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