Single-Molecule Biophysical Assays With Electrokinetics

Brownian dynamics-based estimates of polymer tension are compared with optical traps’ direct force measurements. We suspended phalloidin-stabilized, actin filaments between two beads. The positions of the beads were controlled with optical traps facilitating direct measurement of the forces acting on the beads. The lateral, thermal fluctuations of each filament were visualized with a video camera, and the images were used to obtain the transverse displacement as a function of position along the filament, time, and camera exposure time. The fluctuation’s variance was calculated from the experimental data. When the camera exposure time was increased above a certain threshold, the estimated variance decreased. To obtain an unbiased estimate of the variance, it was necessary to carry out the observations at exposure times shorter than the threshold time interval that was related to the filament’s oscillatory time scale. The variance was estimated as a function of the applied force with a linear Brownian dynamics model. Then, an inverse problem was solved to estimate the filament’s tension. The estimated force was compared and agreed within a factor of 2 with the trap force measurements. The technique described herein can be used for non-contact estimates of polymer’s and fiber’s tension.