SPOTs: Optical Technologies for Instantly Quantifying Multicellular Response Profiles

项目概要/摘要 人体器官系统需要在宏观尺度上时间和空间上协调的多细胞作用， 启动、维持或终止专用和重要功能。包含离散或分布的细胞 生理系统不能协调地对适当的刺激作出反应可能会导致严重的发病率 而且常常是死亡。集体和协调的生理活动通常涉及数百万至数十亿个细胞 可以跨越很大的物理距离。用于量化电、化学和机械性能的技术 这些多细胞系统中的偶联对于理解 疾病和开发治疗方法。然而，目前还没有技术可以量化快速 细胞集合内的所有细胞对传输的远端扰动的机械细胞响应。这多- PI提案（Chiou（联系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），用于在1.0 x 1 1.0 cm 2视场（FOV），具有细胞空间分辨率和1，000帧/秒的时间分辨率。现货 提供比传统牵引力显微镜大4个数量级的FOV。心肌细胞 (CMs)是这里的试验台，因为它对心血管疾病的影响很大， 西方世界的死亡率。我们将展示SPOT确定以下定量指标的能力： 在健康和患病状态下的人CM收缩和舒张的异常。我们会证明 SPOT筛选增强或影响桥粒斑蛋白CM收缩的小分子的概念研究 欠缺的国家。我们将建立一个平台，集成SPOT直接收缩力测量， 光学映射用于CM片材的电性能测量。这将首次使 单胞条件下多层膜的时空机电耦合行为研究 分辨率我们将区分CM的不同亚型，它们的分布，它们的相互作用，以及它们之间的相互作用。 外部扰动下的表型反应。我们将应用这个平台来研究 和机电耦合特性的hESC-衍生的CM通过整合定量的生物质和 使用非侵入性活细胞干涉测量法（LCI）测量的刚度数据。生物量和细胞硬度的变化 是与CM的机械收缩/舒张周期相关的可用药生物物理参数。在 除了对有可能影响美国公民头号杀手的CM进行详细研究外，SPOT 应用程序应该具有实用性，并在需要细胞或组织的其他设置中提供新的见解 牵引力的产生这样的设置可以包括用于伤口愈合、癌细胞培养和细胞培养的培养皿中的模型。 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 Ehlers-Danlos综合征或马凡氏综合征。

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.