SPOTs: Optical Technologies for Instantly Quantifying Multicellular Response Profiles

项目摘要/摘要 人类器官系统需要在时间和空间上协调的大尺度多细胞行动，以 启动、维持或终止专用的和重要的功能。包含离散或分布式的单元格 生理系统不能对适当的刺激做出协调反应，可能会导致严重的疾病 而且常常是死亡。集体和协调的生理活动通常涉及数百万到数十亿个细胞 这可能跨越很长的物理距离。用于量化电气、化学和机械的技术 这些多细胞系统中的耦合对于理解其潜在机制至关重要。 疾病，并开发治疗方法。然而，目前还没有技术来量化快速 一组细胞中的所有细胞对传递的远端扰动的机械反应。这个多- Pi Proposal(Chiou(Contact Pi)和Teitell)旨在开发一种名为SPOT的新平台成像技术 (单像素光学技术)，用于同时和直接测量1.0x以上的蜂窝牵引力 1.0 cm2视场(FOV)，具有蜂窝空间分辨率和1000帧/秒的时间分辨率。斑点 提供比传统牵引力显微镜大4个数量级的视场。心肌细胞 (CMS)是这里的试验台，因为它对心血管疾病的影响潜力很高，心血管疾病是主要原因 西方世界的死亡率。我们将演示SPOT确定定量指标的能力 健康和疾病状态下人体CM收缩和松弛的异常。我们会证明 斑点筛选中增强或影响肌纤维连结蛋白收缩的概念研究 有缺陷的国家。我们将搭建一个集直接收缩测力和Spot于一体的平台 CMS薄片电性测量的光学标测。这将第一次使 单胞CMS薄片的时空机电耦合行为研究 决议。我们将区分不同的CMS亚型，它们的分布，它们的相互作用，以及它们的 外部扰动下的表型反应。我们将利用这个平台来研究结构 和机电耦合特性的hESC衍生的CMS通过整合定量生物量和 使用无创活细胞干涉法(LCI)测量僵硬数据。生物量和细胞硬度的变化 是与CMS的机械收缩/松弛循环相关的可用药的生物物理参数。在……里面 除了对可能影响美国公民头号杀手的CMS的详细研究外，Spot 应用程序应该具有实用性，并在需要细胞或组织的其他环境中提供新的见解 牵引力产生。这样的设置可以包括伤口愈合、癌细胞的培养皿中的模型 转移，或影响细胞和组织结构完整性的疾病模型，如结缔组织 埃勒斯-丹洛综合征或马凡综合征。

PROJECT SUMMARY/ABSTRACT Human organ systems require temporally and spatially coordinated multicellular actions at a macroscale to actuate, sustain, or terminate dedicated and vital functions. Cells that comprise discrete or distributed physiologic systems that fail to respond to appropriate stimuli with coordination may cause significant morbidity and often mortality. Collective and coordinated physiologic activities typically involve millions to billions of cells that may span large physical distances. Technologies for quantifying the electrical, chemical, and mechanical coupling in these multicellular systems are critically important to understanding the underlying mechanisms of disease and develop therapeutic approaches. However, no technology currently exists to quantify rapid mechanical cell responses to transmitted distal perturbations for all cells within a collection of cells. This multi- PI proposal (Chiou (contact PI) and Teitell) aims to develop a new platform imaging technology called SPOT (single pixel optical technology) for concurrent and direct measurements of cellular traction forces over a 1.0 x 1.0 cm2 field of view (FOV) with cellular spatial resolution, and a 1,000 frames/sec temporal resolution. SPOT provides a 4-order of magnitude larger FOV than conventional traction force microscopy. Cardiomyocytes (CMs) are the test bed here because of a high potential for impact in cardiovascular disease, the leading cause of mortality in the Western World. We will demonstrate the ability for SPOT to determine quantitative indices of abnormalities for human CM contraction and relaxation in healthy and diseased states. We will establish proof of concept studies in SPOT screens for small molecules that augment or affect CM contraction in desmoplakin deficient states. We will build a platform that integrates SPOT for direct contraction force measurements and Optical Mapping for electrical property measurements for sheets of CMs. This will enable, for the first time, studies of temporal and spatial electromechanical coupling behaviors for sheets of CMs at single cell resolution. We will distinguish different subtypes of CMs, their distributions, their interactions, and their phenotypic responses under external perturbations. And we will apply this platform to investigate the structural and electromechanical coupling properties of hESC-derived CMs by integrating quantitative biomass and stiffness data measured using non-invasive live cell interferometry (LCI). Changes in biomass and cell stiffness are druggable biophysical parameters with correlates to mechanical contraction/relaxation cycles of CMs. In addition to detailed studies of CMs that have the potential to impact the number one killer of US citizens, SPOT applications should have utility and provide new insights in additional settings that require cell or tissue traction-force generation. Such settings could include models in a dish for wound healing, cancer cell metastasis, or models of diseases that affect cell and tissue structural integrity, such as connective tissue disorders Ehlers-Danlos or Marfan syndromes.