Dual-view Inverted Selective Plane Illumination (diSPIM) Light-sheet Microscopy

利用细胞内动力学获得完整的活体时空张力图 组织 项目摘要 组织中的张力与发育过程中的关键形态变化以及病理变化有关。 引发心血管疾病和癌症转移的转化。普遍采用的技术用于 由于组织的侵袭性，量化细胞张力不适用于组织。这一技术差距是一个关键 生物医学研究障碍，旨在阐明组织力学在疾病进展和治疗中的作用 发育障碍。 我们建议开发一种方法来量化细胞和组织内的张力，方法是 表征主要细胞膜运输途径的张力反应：网状蛋白介导 内吞作用(CME)。我们以前的工作和前兆实验表明，时空变化 通过对细胞内CME动力学的分析，可以确定细胞的张力。我们提出，稳健的 张力和CME动力学之间的反相关关系可以用来发展综合的时空 非侵入性地绘制生命系统的张力图。 在目标1中，我们将描述在细胞张力的不同值下定义CME动力学的参数。我们 还将决定使用CME动力学开发的张力图的空间和时间分辨率。 在目标2中，我们将验证所提出的方法学在活多细胞组织中的适用性。 有机体。我们将以果蝇胚胎发育作为发育的模式生物。在《目标3》中， 我们将开发一个利用CME动力学生成张力图的软件平台。 我们设想，这种高风险、高回报的方法将在生物医学研究方面带来重大飞跃，因为它 提供使用广泛可用的组织的全面时空张力图的非侵入性组装 荧光成像方式。

Utilizing Endocytic Dynamics to Obtain Comprehensive Spatiotemporal Tension Maps of Live Tissues Project Summary Tension in tissues relates to pivotal morphological changes during development, as well as pathologic transformations that trigger cardiovascular disorders and cancer metastasis. Prevalent techniques employed to quantify cellular tension are not applicable to tissues due to their invasive nature. This technical gap is a critical barrier for biomedical research aiming to elucidate the roles of tissue mechanics in disease progression and developmental disorders. We propose developing a methodology for quantifying the tensile forces within cells and tissues by characterizing the tension response of a major cellular membrane traffic pathway: clathrin-mediated endocytosis (CME). Our previous works and precursor experiments show that spatial and temporal variations in tension can be determined through the analysis of CME dynamics in cells. We propose that the robust anticorrelation between tension and CME dynamics can be used to develop comprehensive spatiotemporal tension maps of living systems noninvasively. In Aim 1, we will characterize the parameters defining CME dynamics at distinct values of cell tension. We will also determine the spatial and temporal resolving power of tension maps developed using CME dynamics. In Aim 2, we will validate the applicability of the proposed methodology to tissues of living multicellular organisms. We will use Drosophila melanogaster embryogenesis as the model developing organism. In Aim 3, we will develop a software platform for generating tension maps by utilizing CME dynamics. We envision that this high-risk, high-payoff approach will provide a major leap in biomedical research as it offers noninvasive assembly of comprehensive spatiotemporal tension maps of tissues using broadly available fluorescence imaging modalities.