Plotnikov Sergey V, Sabass Benedikt, Schwarz Ulrich S, Waterman Clare M
National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Institute for Theoretical Physics and BioQuant, Heidelberg University, Heidelberg, Germany.
Methods Cell Biol. 2014;123:367-94. doi: 10.1016/B978-0-12-420138-5.00020-3.
Cellular forces generated by the actomyosin cytoskeleton and transmitted to the extracellular matrix (ECM) through discrete, integrin-based protein assemblies, that is, focal adhesions, are critical to developmental morphogenesis and tissue homeostasis, as well as disease progression in cancer. However, quantitative mapping of these forces has been difficult since there has been no experimental technique to visualize nanonewton forces at submicrometer spatial resolution. Here, we provide detailed protocols for measuring cellular forces exerted on two-dimensional elastic substrates with a high-resolution traction force microscopy (TFM) method. We describe fabrication of polyacrylamide substrates labeled with multiple colors of fiducial markers, functionalization of the substrates with ECM proteins, setting up the experiment, and imaging procedures. In addition, we provide the theoretical background of traction reconstruction and experimental considerations important to design a high-resolution TFM experiment. We describe the implementation of a new algorithm for processing of images of fiducial markers that are taken below the surface of the substrate, which significantly improves data quality. We demonstrate the application of the algorithm and explain how to choose a regularization parameter for suppression of the measurement error. A brief discussion of different ways to visualize and analyze the results serves to illustrate possible uses of high-resolution TFM in biomedical research.
由肌动球蛋白细胞骨架产生并通过离散的、基于整合素的蛋白质组装体（即粘着斑）传递到细胞外基质（ECM）的细胞力，对于发育形态发生、组织稳态以及癌症中的疾病进展至关重要。然而，由于尚无实验技术能够在亚微米空间分辨率下可视化纳牛顿力，因此对这些力进行定量映射一直很困难。在此，我们提供了详细的方案，用于使用高分辨率牵引力显微镜（TFM）方法测量施加在二维弹性底物上的细胞力。我们描述了用多种颜色的基准标记物标记的聚丙烯酰胺底物的制备、用ECM蛋白对底物进行功能化、设置实验以及成像程序。此外，我们提供了牵引力重建的理论背景以及设计高分辨率TFM实验时重要的实验考虑因素。我们描述了一种用于处理在底物表面下方拍摄的基准标记物图像的新算法的实现，该算法显著提高了数据质量。我们展示了该算法的应用，并解释了如何选择正则化参数以抑制测量误差。对可视化和分析结果的不同方法的简要讨论有助于说明高分辨率TFM在生物医学研究中的可能用途。
