Carlucci Laura A, Thomas Wendy E
Department of Bioengineering, University of Washington, Seattle, Washington, 98195, USA.
Biophys Rep (N Y). 2021 Dec 8;1(2). doi: 10.1016/j.bpr.2021.100031. Epub 2021 Nov 10.
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.
三维粒子跟踪是包括磁镊在内的各种生物物理应用中的常规实验程序。跟踪粒子轴向位置的一种常用方法是分析衍射环，其图案敏感地取决于珠子相对于焦平面的轴向位置。为了推断轴向位置，将观察到的环与珠子在已知轴向位置的参考图像进行比较。这些算法的精度或准确性通常是在有限轴向范围内对固定珠子进行测量的，而许多实验是使用自由移动的珠子进行的。这种不一致增加了对实验不确定性估计错误的可能性。通过在双向磁镊装置中操纵磁珠，我们评估了跟踪移动磁珠相关的误差，发现跟踪移动磁珠的误差与跟踪固定磁珠的误差相比增加了近一个数量级。我们发现，通过在跟踪分析中排除衍射环图案的最中心区域，可以改善这种额外的误差。评估跟踪算法的局限性对于理解与测量相关的误差至关重要。这些发现有望提高磁性微球三维珠子跟踪的分辨率。
