High-resolution traction force microscopy on small focal adhesions - improved accuracy through optimal marker distribution and optical flow tracking.
High-resolution traction force microscopy on small focal adhesions - improved accuracy through optimal marker distribution and optical flow tracking.
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DOI：
10.1038/srep41633
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发表时间：
2017-02-06
影响因子：
4.6
通讯作者：
Snedeker JG
中科院分区：
文献类型：
--
作者：
Holenstein CN;Silvan U;Snedeker JG
使用牵引力显微镜在与细胞外环境的越来越小的焦点附着处准确测定细胞力提出了重要但实质性的技术挑战。在这些测量中，关于准确性的不确定性是突出的，因为在这种规模的实验校准框架充满了错误-否认了可以判断TFM方法准确性的金标准。因此，我们已经开发了一个模拟平台，用于生成合成牵引图像，可以用作基准，以量化关键实验参数和相关误差的影响。使用这种方法，我们表明，与标准方法相比，通过将荧光珠尽可能密集和靠近施加牵引的部位，TFM准确性可以提高>35%。此外，我们使用该平台来测试基于光流的跟踪算法，这些算法直接在珠子上测量变形，并表明这些算法在噪声敏感性和误差方面可以大大优于经典的粒子图像测速算法。然后，我们报告如何优化的实验和数值策略可以提高牵引力地图的准确性，并进一步提供最好的基准，以确定实际的限制TFM的准确性作为一个功能的焦点粘附大小。
The accurate determination of cellular forces using Traction Force Microscopy at increasingly small focal attachments to the extracellular environment presents an important yet substantial technical challenge. In these measurements, uncertainty regarding accuracy is prominent since experimental calibration frameworks at this size scale are fraught with errors – denying a gold standard against which accuracy of TFM methods can be judged. Therefore, we have developed a simulation platform for generating synthetic traction images that can be used as a benchmark to quantify the influence of critical experimental parameters and the associated errors. Using this approach, we show that TFM accuracy can be improved >35% compared to the standard approach by placing fluorescent beads as densely and closely as possible to the site of applied traction. Moreover, we use the platform to test tracking algorithms based on optical flow that measure deformation directly at the beads and show that these can dramatically outperform classical particle image velocimetry algorithms in terms of noise sensitivity and error. We then report how optimized experimental and numerical strategy can improve traction map accuracy, and further provide the best available benchmark to date for defining practical limits to TFM accuracy as a function of focal adhesion size.