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Optical tweezers in single-molecule biophysics

Nature Reviews Methods Primers volume 1, Article number: 25 (2021) Cite this article

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Abstract

Optical tweezers have become the method of choice in single-molecule manipulation studies. In this Primer, we first review the physical principles of optical tweezers and the characteristics that make them a powerful tool to investigate single molecules. We then introduce the modifications of the method to extend the measurement of forces and displacements to torques and angles, and to develop optical tweezers with single-molecule fluorescence detection capabilities. We discuss force and torque calibration of these instruments, their various modes of operation and most common experimental geometries. We describe the type of data obtained in each experimental design and their analyses. This description is followed by a survey of applications of these methods to the studies of protein–nucleic acid interactions, protein/RNA folding and molecular motors. We also discuss data reproducibility, the factors that lead to the data variability among different laboratories and the need to develop field standards. We cover the current limitations of the methods and possible ways to optimize instrument operation, data extraction and analysis, before suggesting likely areas of future growth.

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Fig. 1: Principles of optical tweezers.
Fig. 2: Basic designs of optical traps.
Fig. 3: Measurement geometries of standard optical traps, fleezers and angular optical traps.
Fig. 4: Example optical trapping data.
Fig. 5: Example fluorescence-trap data.
Fig. 6: Example angular optical tweezers data.
Fig. 7: Example applications of optical tweezers to study protein–DNA interactions.
Fig. 8: Example applications of optical tweezers to study protein folding.
Fig. 9: Example applications of optical tweezers to study molecular motors.

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Acknowledgements

The authors dedicate this Primer to A. Ashkin (1922–2020), whose ingenious invention of optical tweezers has greatly influenced the field of single-molecule biophysics. This work is supported by the Nanomachines programme (KC1203) funded by the Office of Basic Energy Sciences of the US Department of Energy (DOE) contract no. DE-AC02-05CH11231, National Institutes of Health (NIH) grants R01GM032543 (to C.J.B.), R01GM120353 (to Y.R.C.), DP2HG010510 (to S.L.) and R01GM136894 (to M.D.W.), and National Science Foundation (NSF) grants PHY-1430124 (Physics Frontiers Center (PFC) ‘Center for the Physics of Living Cells’ to Y.R.C.) and MCB-1517764 (to M.D.W.). C.J.B. and M.D.W. are Howard Hughes Medical Institute investigators. S.L. is supported by the Robertson Foundation and thanks M. Wasserman for discussions. M.D.W. thanks J. Inman and G. Xiang for commenting on the manuscript.

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Carlos J. Bustamante

  2. Department of Physics, University of California, Berkeley, CA, USA

    Carlos J. Bustamante

  3. Department of Chemistry, University of California, Berkeley, CA, USA

    Carlos J. Bustamante

  4. Kavli Energy NanoScience Institute, University of California, Berkeley, CA, USA

    Carlos J. Bustamante

  5. Howard Hughes Medical Institute, University of California, Berkeley, CA, USA

    Carlos J. Bustamante

  6. Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Yann R. Chemla

  7. Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, New York, NY, USA

    Shixin Liu

  8. Department of Physics, Laboratory of Atomic and Solid State Physics, Howard Hughes Medical Institute, Cornell University, Ithaca, NY, USA

    Michelle D. Wang

Authors
  1. Carlos J. Bustamante
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  2. Yann R. Chemla
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  3. Shixin Liu
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  4. Michelle D. Wang
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Contributions

Introduction (C.J.B.); Experimentation (Y.R.C. and M.D.W.); Results (S.L., Y.R.C. and M.D.W.); Applications (M.D.W., C.J.B., S.L. and Y.R.C.); Reproducibility and data deposition (S.L.); Limitations and optimizations (Y.R.C.); Outlook (C.J.B.); Overview of the Primer (C.J.B.).

Corresponding authors

Correspondence to Carlos J. Bustamante, Yann R. Chemla, Shixin Liu or Michelle D. Wang.

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The authors declare no competing interests.

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Nature Reviews Methods Primers thanks Yuxuan Ren, Felix Ritort, Sander Tans and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Glossary

Thermal bath

(Also known as heat bath, or thermal reservoir in thermodynamics and statistical mechanics). A large body held at constant temperature in which the system of interest is immersed and with which it is in thermal equilibrium. It is assumed to possess a large heat capacity and to be essentially an infinite source of thermal energy so that it can exchange energy with the system (as a thermal source or as a heat sink) without altering its temperature.

Torques

Cross products of the position vector (pointing from the axis of rotation to the point of application of the force) and the force driving the rotation.

Wavelength

The distance between two maxima of the light’s electric or magnetic field.

Rayleigh scatterer

An object whose dimensions are much smaller than the wavelength of the light.

Polarizability

A dipole moment is generated when a distribution of charges experiences a separation of positive and negative charges under the influence of an electric field. The magnitude of the dipole is proportional to the amplitude of the electric field \(\mathop{E}\limits^{\rightharpoonup }\). The constant of proportionality is the polarizability of the charge distribution.

Electric dipole

A pair of electric charges of equal magnitude but opposite signs separated by a finite distance.

Gaussian laser beam

A light beam whose intensity can be described with a Gaussian function, maximal at its axis and decreasing towards its periphery.

Permittivity

A measure of the electric polarizability of the medium.

Absorption

A process in which the energy carried by light is transferred to the sample.

Refraction

A change in the direction of the travelling beam by the presence of an object that has a different index of refraction to the surrounding medium, when that beam impinges on it obliquely.

Reflection

A process by which the light that impinges on a surface of an object bounces back from that surface instead of being absorbed or refracted by the object.

Extinction cross-section

A measure of the efficiency with which a given object can absorb or scatter the light that impinges on it.

Poynting vector

A quantity that describes the magnitude and direction of the flow of energy of a propagating electromagnetic wave.

Geometric or ray optics

A description of light in terms of rays that describe in an approximate manner the paths along which the light travels.

Refractive objects

Objects that possess a different index of refraction to the medium in which they are immersed and that deflect light that impinges on them obliquely.

High numerical aperture

The range of angles over which a lens can collect light; the larger the numerical aperture, the larger the angular range.

Condenser lens

A standard optical microscope component typically used to focus the microscope light into the specimen; in optical traps it is often used to collect scattered trap light.

Back focal plane interferometry

A technique in which the interference pattern between the light transmitted and the light forward-scattered by the trapped particle is used to determine the position of the particle relative to the trap centre.

Linear range

The range of input parameters over which the output of the system depends linearly on the input.

Measurement bandwidths

The rates at which data are collected.

Boltzmann constant

A fundamental physical constant that relates the kinetic energy of a gas to its absolute temperature. It is closely related to the gas constant R.

Hydrodynamic drag coefficient

A coefficient characterizing the viscous resistance that a particle moving through a fluid encounters.

Pointing drift

Unwanted changes in the direction of a beam of light.

Brownian motion

The random fluctuation of a particle’s position (and orientation) at a given temperature.

Feedback loop

A self-regulating mechanism in which the output of a system is routed back into an input to the system. In negative feedback, the output is fed back in a fashion that tends to reduce fluctuations in the output.

Fluorophores

Fluorescent chemical compounds or molecules.

Oxygen scavengers

Enzymatic systems that remove molecular oxygen detrimental to fluorescence.

Extraordinary axis

(Also known as the ‘optic axis’). For a uniaxial crystal that has a refractive index of one crystal axis that is different from the other two crystal axes, light propagating parallel to this optic axis experiences the same index of refraction regardless of its polarization.

Laser polarization

The polarization of the light refers to the direction of the electric field.

Laguerre–Gaussian beam

A mode of laser that can carry both orbital and spin angular momentum.

Michaelis–Menten kinetic parameters

Parameters that relate the rate of an enzymatic reaction v to the substrate concentration [S] by the formula v = Vmax[S] / (KM + [S]), where Vmax and KM represent the maximum rate and the substrate concentration at which the reaction rate is half of Vmax, respectively.

Temporal resolution

The measure of the fastest signal that can be detected accurately.

Biolistic

A method of introducing beads inside cells by shooting them ballistically inside the cells at supersonic speeds.

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Bustamante, C.J., Chemla, Y.R., Liu, S. et al. Optical tweezers in single-molecule biophysics. Nat Rev Methods Primers 1, 25 (2021). https://doi.org/10.1038/s43586-021-00021-6

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