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Multiplexed Nanometric 3D Tracking of Microbeads using an FFT-Phasor Algorithm
Biophysical Journal ( IF 3.4 ) Pub Date : 2020-05-01 , DOI: 10.1016/j.bpj.2020.01.015
Thomas B Brouwer 1 , Nicolaas Hermans 1 , John van Noort 1
Affiliation  

Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead’s position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 μm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.

中文翻译:

使用 FFT 相量算法对微珠进行多重纳米 3D 跟踪

许多单分子生物物理技术依靠微珠的纳米跟踪来获得有关生物分子(如染色质纤维)机械性能的定量信息。它们的三维 (3D) 位置可以通过对宽视场成像中的衍射图案进行全息分析来解决。将这种衍射图案与洛伦兹-米散射理论拟合,可以在三个维度上以纳米精度产生珠子的位置,但计算成本很高。因此,实时多路复用珠跟踪需要更有效的跟踪方法,例如与先前测量的衍射图案进行比较,称为查找表。这里,我们引入了一种替代的 3D 相量算法,该算法在非最佳成像条件下提供了 az 范围内超过 10 μm 的纳米定位精度的稳健珠跟踪。该算法基于二维互相关,使用计算机生成的参考图像进行快速傅立叶变换,产生高达每秒 10,000 个感兴趣区域的处理速率。我们在磁性镊子中实施了该技术,并在通用 CPU 上实时跟踪了 100 多个珠子的 3D 位置。3D 相量跟踪的准确性经过了广泛的测试,并与使用 Lorenz-Mie 模拟的查找表方法进行了比较,从而避免了实验的不确定性。其易于实施、效率和稳健性可以改善多路生物物理珠跟踪应用,
更新日期:2020-05-01
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