Brouwer Thomas B, Hermans Nicolaas, van Noort John
Biological and Soft Matter Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, the Netherlands.
Biological and Soft Matter Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, the Netherlands.
Biophys J. 2020 May 5;118(9):2245-2257. doi: 10.1016/j.bpj.2020.01.015. Epub 2020 Jan 23.
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.
许多单分子生物物理技术依靠对微珠的纳米级追踪来获取有关生物分子（如染色质纤维）机械特性的定量信息。通过对宽场成像中衍射图案的全息分析，可以解析它们的三维（3D）位置。将此衍射图案与洛伦兹 - 米氏散射理论拟合，可在三维空间中以纳米精度得出珠子的位置，但计算成本很高。因此，实时多路复用珠子追踪需要一种更高效的追踪方法，例如与先前测量的衍射图案进行比较，即查找表。在这里，我们介绍一种替代的3D相量算法，该算法在非最佳成像条件下，在超过10μm的z范围内，能以纳米级定位精度提供稳健的珠子追踪。该算法基于使用快速傅里叶变换和计算机生成的参考图像进行二维互相关，每秒可处理多达10,000个感兴趣区域。我们在磁镊中实现了该技术，并在通用CPU上实时追踪了100多个珠子的3D位置。通过洛伦兹 - 米氏模拟对3D相量追踪的准确性进行了广泛测试，并与查找表方法进行了比较，避免了实验不确定性。其易于实现性、效率和稳健性可改善多路复用生物物理珠子追踪应用，特别是在需要高通量且难以避免图像伪影的情况下。
