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 experimentBN/BionanoscienceApplied Science

Quantitative Guidelines for Force Calibration through Spectral Analysis of Magnetic Tweezers Data

Single-molecule techniques are powerful tools that can be used to study the kinetics and mechanics of a variety of enzymes and their complexes. Force spectroscopy, for example, can be used to control the force applied to a single molecule and thereby facilitate the investigation of real-time nucleic acid-protein interactions. In magnetic tweezers, which offer straightforward control and compatibility with fluorescence measurements or parallel tracking modes, force-measurement typically relies on the analysis of positional fluctuations through video microscopy. Significant errors in force estimates, however, may arise from incorrect spectral analysis of the Brownian motion in the magnetic tweezers. Here we investigated physical and analytical optimization procedures that can be used to improve the range over which forces can be reliably measured. To systematically probe the limitations of magnetic tweezers spectral analysis, we have developed a magnetic tweezers simulator, whose outcome was validated with experimental data. Using this simulator, we evaluate methods to correctly perform force experiments and provide guidelines for correct force calibration under configurations that can be encountered in typical magnetic tweezers experiments