Non-bias-limited tracking of spherical particles, enabling nanometer resolution at low magnification.

We present a three-dimensional tracking routine for nondiffraction-limited particles, which significantly reduces pixel bias. Our technique allows for increased resolution compared to that of previous methods, especially at low magnification or at high signal/noise ratio. This enables tracking with nanometer accuracy in a wide field of view and tracking of many particles. To reduce bias induced by pixelation, the tracking algorithm uses interpolation of the image on a circular grid to determine the x-, y-, and z-positions. We evaluate the proposed algorithm by tracking simulated images and compare it to well-known center-of-mass and cross-correlation methods. The final resolution of the described method improves up to an order of magnitude in three dimensions compared to conventional tracking methods. We show that errors in x,y-tracking can seriously affect z-tracking if interpolation is not used. We validate our results with experimental data obtained for conditions matching those used in the simulations. Finally, we show that the increased performance of the proposed algorithm uniquely enables it to extract accurate data for the persistence length and end-to-end distance of 107 DNA tethers in a single experiment.