Asymmetric Illumination of Optically Anisotropic Beads for Detecting Rotational Motion

Abstract

Many biological molecules, including RNA polymerases and ATP synthases, undergo rotational motion during their biological processes. Characterizing these processes at the single molecule level with high temporal and spatial resolution can reveal valuable information about their mechanical and dynamical properties. We are particularly interested in the dynamics of conformational changes of DNA molecules under torsion. Toward this long term goal we have developed a methodology that enables us to directly record the angular displacement of particles undergoing rotational motion by using asymmetric illumination of optically anisotropic beads. The method is implemented by illuminating a partially metal-coated bead with a laser beam coupled into the back side of the objective. The laser beam illuminating beads in the sample plane is oriented at an oblique angle to form asymmetric illumination. We observe that the scattering signal of the bead changes with the angular displacement of the coating on the bead relative to the illuminating laser beam. We use an optoelectronic system to detect scattering signal which is the direct high-speed measurement of the angular displacement of the bead. We conclude that our method is able to map angular displacements to electrical signal and we can determine the angular displacement of the biological molecule when conjugated to the bead. Our method obviates image acquisition and image processing procedures commonly used in previous studies, and it has the potential to significantly enhance the bandwidth of detection. We envision usage of this method in a range of biophysical measurements including magnetic trapping, tethered particle motion. We plan to use this rotational tracking method in combination with magnetic tweezers, a single-molecule technique that enables the application of torsional stress to twist single DNA molecules to extract high bandwidth torsional mechanical properties and dynamics of the molecules.