A Novel Method for Multiplexed Nanometric Bead Tracking

Abstract

Single-molecule force spectroscopy is a powerful tool to resolve dynamics and interactions between large bio-molecules. Magnetic Tweezers, for example, probe the mechanics of a biopolymer by tethering it to a micrometer sized bead and tracking the bead using video microscopy and image processing. This technique strongly relies on accurate bead tracking in three dimensions with nanometer resolution. However, it traditionally suffers from extremely low throughput. When parallelizing bead-tracking experiments, the need arises for fast algorithms without compromising accuracy and robustness. Here, we introduce a novel tracking method based on a 3D cross correlation of the bead image with a set of computer-generated reference images. We show that the phase shift of the cross correlation peak is proportional to the bead height. For testing the accuracy, we used Lorentz-Mie scattering theory to generate test data. We found that non-circular effects such as interlacing, small image artifacts and non-uniform illumination do not affect the tracking result. Our algorithm tracks over 100 beads in a 100x100 pixel region-of-interest on a multicore PC in real time. Its speed, accuracy and robustness may improve other techniques that rely on bead tracking such as optical tweezers and acoustic force spectroscopy.