Optical tweezers in single-molecule biophysics (Bustamante et al., Nat Rev Methods Primers 2021)
Optical tweezers have become arguably the method of choice in single-molecule manipulation studies. Writing with three leaders in the field, 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 measurements of forces and displacements to torques and angles, and to integrate them with single-molecule fluorescence detection modalities. We discuss force and torque calibration of these instruments, their various modes of operation and most common experimental geometries. We describe the types of data obtained in each experimental design and the common ways to analyze them, 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 areas of future growth.