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

利用内吞动力学获得完整的活细胞时空张力图 组织 项目摘要 组织中的张力与发育过程中的关键形态学变化以及病理学变化有关。 这些转化会引发心血管疾病和癌症转移。采用的流行技术， 量化细胞张力由于其侵入性而不适用于组织。这一技术差距是一个关键 生物医学研究的屏障，旨在阐明组织力学在疾病进展中的作用， 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， 表征主要细胞膜运输途径的张力反应：网格蛋白介导的 胞吞作用（CME）。我们以前的工作和先驱实验表明，空间和时间变化 可以通过分析细胞中的CME动力学来确定张力。我们建议， 张力与日冕物质抛射动力学之间的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，我们将描述在不同细胞张力值下定义CME动力学的参数。我们 还将确定使用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对活的多细胞组织的适用性。 有机体我们将使用果蝇胚胎发生作为模式发育生物。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、高回报的方法将为生物医学研究提供一个重大飞跃，因为它 提供了使用广泛可用的组织的全面时空张力图的非侵入性组装 荧光成像模式。

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.