Innovative Tools for Three Dimensional Traction Force Microscopy of Single Cells

项目摘要 单个细胞对其周围环境施加的牵引力在生物学中的机械事件中起着关键作用 例如组织收缩、折叠、细胞形状改变或细胞运动，且在许多基本细胞中 具有生化信号、增殖和分化等功能。这些过程依次为 与癌症、动脉粥样硬化和其他慢性纤维化等疾病的进展有关。 最近，这种显著的联系已被用于开发令人兴奋的新治疗干预措施，这些干预措施依赖于 扰乱细胞内的机械信号机制，以及导致细胞重塑的途径 细胞外基质(ECM)。 可以精确量化细胞牵引力的空间变化和异质性的技术 细胞之间的相互作用在理解和控制这些过程中发现了重要的应用。其中， 三维牵引力显微镜(3DTFM)已经成为一种特别有价值的工具，因为它是 应用于嵌入细胞的三维ECM，这是大多数细胞的自然状态。当前的3D TFM 使用光学图像生成3D几何模型的关键步骤对方法提出了挑战 细胞周围的基质，并从微珠的位移估计推断细胞牵引力 嵌入在矩阵中。在这些步骤中产生的近似会导致计算的牵引力出现显著误差 这反过来又导致了错误的生物学结论。因此，迫切需要开发出更准确和 高分辨率3D TFM技术。 拟议研究的长期目标是改进3D TFM过程并使其自动化，从而使其 可以有效地用于回答机械生物学问题和设计新的治疗干预措施。这 将通过(A)将先进的分割和网格生成技术应用于光学 图像以生成围绕单元格的矩阵的3D几何模型和有限元网格，以及(B) 通过开发和实施新的算法来确定细胞牵引力的空间分布 测量了微珠的位移，同时考虑了基质的非线性弹性响应。这些 发展将通过基准研究来验证，其效用将通过量化来展示 癌细胞嵌入到合成的细胞外基质中所施加的牵引力。

Project Summary Tractions exerted by individual cells on their surroundings play a critical role in mechanical events in biology such as tissue contraction, folding, cell shape changes, or cell movements, and in many basic cellular functions such as biochemical signaling, proliferation, and differentiation. These processes are in turn implicated in the progression of diseases like cancer, atherosclerosis, and other chronic fibrotic conditions. Recently, this remarkable link has been utilized to develop exciting new therapeutic interventions that rely on disrupting mechano-signaling machinery within the cell, and the pathways that lead to the remodeling of the extra-cellular matrix (ECM). Techniques that can precisely quantify the spatial variation and heterogeneity of cellular traction within and between cells have found important applications in understanding and controlling these processes. Of these, three-dimensional traction force microscopy (3D TFM) has emerged as a particularly valuable tool since it is applied to cells embedded in a three-dimensional ECM, the natural state for most cells. Current 3D TFM approaches are challenged by the critical steps of using optical images to generate a 3D geometrical model of the matrix surrounding the cell, and inferring cellular tractions from displacement estimates of micro-beads embedded in the matrix. Approximations incurred in these steps lead to significant errors in computed tractions that in turn lead to erroneous biological conclusions. Thus there is critical need to develop more accurate and high resolution 3D TFM techniques. The long-term objective of the proposed research is to improve and automate the 3D TFM process so that it can be effectively used to answer mechanobiological questions and design new therapeutic interventions. This will be accomplished by (a) applying advanced segmentation and mesh generation techniques to optical images to generate 3D geometric models and finite element meshes of the matrix surrounding a cell, and (b) by developing and implementing new algorithms to determine the spatial distribution of cellular tractions from measured micro-beads displacements, while accounting the nonlinear elastic response of the matrix. These developments will be validated through benchmark studies, and their utility will be demonstrated by quantifying the traction exerted by cancer cells embedded in a synthetic extracellular matrix.