Abstract
Three-dimensional particle tracking is a routine experimental procedure for various biophysical applications including magnetic tweezers. A common method for tracking the axial position of particles involves the analysis of diffraction rings whose pattern depends sensitively on the axial position of the bead relative to the focal plane. To infer the axial position, the observed rings are compared with reference images of a bead at known axial positions. Often the precision or accuracy of these algorithms is measured on immobilized beads over a limited axial range, while many experiments are performed using freely mobile beads. This inconsistency raises the possibility of incorrect estimates of experimental uncertainty. By manipulating magnetic beads in a bidirectional magnetic tweezer setup, we evaluated the error associated with tracking mobile magnetic beads and found that the error of tracking a moving magnetic bead increases by almost an order of magnitude compared to the error of tracking a stationary bead. We found that this additional error can be ameliorated by excluding the center-most region of the diffraction ring pattern from tracking analysis. Evaluation of the limitations of a tracking algorithm is essential for understanding the error associated with a measurement. These findings promise to bring increased resolution to three-dimensional bead tracking of magnetic microspheres.