Dynamics in Tethered Particle Motion: Interpreting the Observations

Abstract

Tethered Particle Motion (TPM) enables the researcher to examine the properties of semi-flexible polymers at the single molecule level. In TPM, a small reporter particle is tethered to a substrate using the polymer of interest. The particle motion reflects the mechanical properties of the tethering polymer.We will present a framework in which the influence of the different experimental aspects on the measurement outcome are quantified. The key elements are tether length, particle size, exposure time, fluid properties and frame rate.Here, we use 80 nanometer diameter gold particles, tethered to a glass slide by double stranded DNA, that are visualized by dark field microscopy. The recorded images of these highly scattering particles have a high contrast and signal-to-noise ratio; therefore the particle can be tracked with high spatial resolution (5-20 nm).High temporal resolution is necessary to distinguish between different diffusion regimes. At very short time scales the particle is freely diffusing and on longer time scales its motion is influenced and eventually restricted by the retracting harmonic force of the tether. We will show that the diffusion coefficient of the free motion on short time scales is composed of the diffusion properties of both the particle and tether and that the harmonic potential stems from the entropic elasticity of the tether molecule. Motion blur caused by the finite exposure time has to be considered for computing the diffusion constant. Lastly, the choice of tether length and particle size play an important role as well. They determine how often the particle will be in proximity of the substrate where particle-substrate interactions such as van-der-Waals and electrostatic forces play a bigger role.