Optical Pushing: A Tool for Parallelized Biomolecule Manipulation

Abstract

The ability to measure and manipulate single molecules has greatly advanced the field of biophysics. Yet, the addition of more single-molecule tools that enable one to measure in a parallel fashion is important to diversify the questions that can be addressed. Here we present optical pushing (OP), a single-molecule technique that is used to exert forces on many individual biomolecules tethered to microspheres using a single collimated laser beam. Forces ranging from a few femtoNewtons to several picoNewtons can be applied with a submillisecond response time. To determine forces exerted on the tethered particles by the laser, we analyzed their measured Brownian motion using, to our knowledge, a newly derived analytical model and numerical simulations. In the model, Brownian rotation of the microspheres is taken into account, which proved to be a critical component to correctly determine the applied forces. We used our OP technique to map the energy landscape of the protein-induced looping dynamics of DNA. OP can be used to apply loading rates in the range of 10(-4)-10(6) pN/s to many molecules at the same time, which makes it a tool suitable for dynamic force spectroscopy.

Faster Uphill Relaxation in Thermodynamically Equidistant Temperature Quenches

“…Implying that the hot isothermal step can be shorter than the cold one, which reduces cycle times, the asymmetry may also be relevant for the optimization of the engine's output power [44,76,77]. Our results can readily be tested by single-molecule and particle-tracking experiments [4,[44][45][46][47][48]. To understand the asymmetry on the level of individual trajectories, it would be interesting to analyze relaxation from equidistant quenches in terms of occupation measures [7,8] and from the perspective of stochastic thermodynamics [12,14].…”

“…It is meanwhile possible to probe the transient, nonequilibrium dynamics of colloids and single molecules, e.g., by temperature-modulated particle tracking [4] and timemodulated [44], temperature-modulated [45], temperaturejump [46], and holographic [47] optical tweezers, as well as optical pushing [48]. These experiments allow for systematic investigations of the dependence of relaxation on the direction of the displacement from equilibrium, which is the central question of the present Letter.…”

Optical manipulation from the microscale to the nanoscale: fundamentals, advances and prospects

“…In the past two decades, a variety of biological mechanisms have been investigated using optical tweezers, such as the dynamics of motor molecules 165 , the motion of RNA polymerase during transcription ( Figure 7a ), the motion of ribosomes during translation 170 , protein folding 171 and DNA-protein binding. Optical tweezers can also be used to trap and study single cells and organelles within cells 172 , 173 , 174 . However, fixed optical tweezers have some restrictions: they can only apply limited forces of 0.1–100 pN and can measure a range of motion of ~400 nm or less 175 .…”

A Wi-Fi live streaming Centrifuge Force Microscope for benchtop single-molecule experiments

“…Since the initial development of the CFM, there have been several other techniques that were developed to provide multiplexed single-molecule experimentation. In particular, there have been significant advances in multiplexed magnetic tweezers (13,14) as well as development of new technologies like acoustic force spectroscopy (AFS) (15) and optical pushing (16). These methods have helped expand the field, especially with commercialization of AFS, but we still believe that the CFM offers some distinct features for ease of use, low cost, and a wide and calibration-free force range.…”