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  • Review Article
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Single-molecule FRET for probing nanoscale biomolecular dynamics

Nature Reviews Physics volume 6, pages 587–605 (2024)Cite this article

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Abstract

Single-molecule spectroscopy is a powerful method for studying the physics of molecular systems, particularly biomolecules, such as proteins and nucleic acids. By avoiding ensemble averaging, single-molecule techniques can resolve structural distributions and fluctuations even for complex and conformationally heterogeneous systems; they also reveal the close link between biological function and the statistical mechanics of the underlying processes. The combination of single-molecule fluorescence detection with Förster resonance energy transfer has become an essential tool for probing biomolecular dynamics on timescales ranging from nanoseconds to days. This Review briefly outlines the state of the art of single-molecule Förster resonance energy transfer spectroscopy and then highlights some of the most important physics-based developments that are expected to further expand the scope of the technique. Key areas of progress include improved time resolution, access to nonequilibrium dynamics and synergies with advances in data analysis and simulations. These developments create new opportunities for attaining a comprehensive understanding of the dynamics and functional mechanisms of biological processes at the nanoscale.

Key points

  • The functions of biological macromolecules depend on changes in their conformations across 24 orders of magnitude in time.

  • Single-molecule Förster resonance energy transfer can be used to probe biomolecular dynamics on nanometre-length scales across timescales from nanoseconds to days.

  • An important challenge is to increase the time resolution for measurements of rapid dynamics and nonequilibrium processes.

  • Nanophotonics, microfluidic mixing and advances in data analysis and molecular simulations are particularly promising strategies for extending the scope of single-molecule Förster resonance energy transfer.

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Fig. 1: Timescales of biomolecular dynamics probed with single-molecule Förster resonance energy transfer.
Fig. 2: Nanophotonic enhancement of fluorescence for single-molecule spectroscopy.
Fig. 3: Nonequilibrium single-molecule dynamics.
Fig. 4: Data analysis and simulations.

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Acknowledgements

The authors thank R. Covino, S. Gopi, G. Haran, H. Hofmann, E. Lipman, C. Lorenz and D. Makarov for insightful discussions and comments on the manuscript. This work was supported by the Swiss National Science Foundation and the Forschungskredit of the University of Zurich.

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Authors and Affiliations

  1. Department of Biochemistry, University of Zurich, Zurich, Switzerland

    Daniel Nettels, Nicola Galvanetto, Miloš T. Ivanović, Mark Nüesch, Tianjin Yang & Benjamin Schuler

  2. Department of Physics, University of Zurich, Zurich, Switzerland

    Nicola Galvanetto & Benjamin Schuler

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Glossary

Bayesian nonparametrics

A type of statistical models and methods characterized by large parameter spaces, such as unknown numbers of microstates and their connectivity, and by the construction of probability measures over these spaces.

Chemical kinetics

Description of the time dependence of the interconversion between thermodynamic states and microstates of a system in terms of rates.

Coarse-grained

In modelling complex systems or in renormalization, coarse-graining refers to the procedure in which two or more microscopic entities are replaced with a single entity to reduce the complexity or resolution of the model.

Droplet microfluidics

A method to manipulate discrete, typically picolitre volumes of fluids in immiscible phases. For biomolecules, aqueous droplets in oil are commonly used.

Ensemble average

The mean value of some observables obtained from simultaneous measurements of all members of a statistical ensemble. Single-molecule spectroscopy overcomes ensemble averaging.

Fluorescence correlation spectroscopy

(FCS). Statistical analysis of fluctuations in fluorescence intensity or count rates via time correlation. FCS is a broadly applicable way of assessing biomolecular dynamics over a broad range of timescales.

Förster resonance energy transfer

(FRET). Non-radiative transfer of excitation energy between two molecular entities separated by distances considerably exceeding the sum of their van der Waals radii in the very weak dipole–dipole coupling limit.

Hydrodynamic focusing

A technique used in microfluidics, in which several fluid streams are combined in microfluidic channels to form a layer or jet that is so thin that it exchanges its solutes very rapidly with the neighbouring streams by diffusion.

Local density of optical states

Measures the availability of electromagnetic modes at a given point in space and governs the deexcitation of a quantum emitter.

Multiparameter fluorescence detection

Simultaneous acquisition of multiple fluorescence observables, such as wavelength, count rate, lifetime and anisotropy, as a function of time in a single measurement.

Nanosecond FCS

(ncFCS). Variant of FCS that enables dynamics in the submicrosecond range to be measured by using a Hanbury Brown and Twiss configuration of single-photon detectors.

Photon antibunching

Special distribution of time delays between photons that is characteristic for the emission of a single quantum emitter. Photon antibunching is detected as an anticorrelated component in fluorescence correlation spectroscopy on timescales comparable to the fluorescence lifetime.

Reaction coordinate

A quantity used to describe the progress of a reaction, often chosen to reflect a change in experimental signal. In the context of Förster resonance energy transfer experiments, the reaction coordinate would typically be related to an intramolecular or intermolecular distance change.

Reconfiguration times

Relaxation time of the correlation function of a point-to-point distance within a molecule, most commonly a polymer chain.

Simulation-based inference

Emerging family of methods that infer the model parameters when the likelihood is intractable by integrating simulations with machine learning.

Single-molecule spectroscopy

Methods that enable the physical properties of individual molecules to be measured.

Time average

The mean value of some observables obtained from measurements of an individual member of the ensemble as a function of time, for example, as a result of time binning. Single-molecule spectroscopy overcomes time averaging for processes that can be resolved with the time resolution of the specific measurement.

Transition paths

The successful reactant-to-product crossing of the free-energy barrier separating two free-energy minima. Transition paths are rare events with very short duration and thus challenging to resolve experimentally.

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Nettels, D., Galvanetto, N., Ivanović, M.T. et al. Single-molecule FRET for probing nanoscale biomolecular dynamics. Nat Rev Phys 6, 587–605 (2024). https://doi.org/10.1038/s42254-024-00748-7

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