3D Quantitative Fluorescent Speckle Microscopy

摘要 荧光散斑显微镜(FSM)是一种可视化和量化的成像模式 活细胞中大分子组装的动力学。它依赖于极亚化学计量比 对感兴趣组件的一个或多个组件进行标记。当通过绕射成像时 有限光学这种标记生成随机的点状纹理，该纹理以统计的形式编码 时尚运输，机械变形，和分子的组装周转。因此， FSM与超分辨率技术Storm和Palm有关，这两种技术也都依赖于 大分子组件的分子组成的随机抽样。在暴风雨中 在标记中，通过被动或主动切换一小组 荧光探头介于暗状态和亮状态之间。相比之下，在最初的实现中 FSM依赖于一组永久标记的亚基，这些亚基动态地合并到 程序集。在《暴风雨》和《棕榈树》中，利用亚化学计量学标记来顺序地 以纳米级精度收集单个亚单位的坐标，即随时间获取 组件的分子组织的超分辨率地图。在FSM中，亚化学计量学 利用标记技术对子单元的运动、添加和移除进行实时跟踪。因此， FSM的空间分辨率仍然受衍射限制；然而，FSM提供了有关 其他成像方式无法提供的大分子组装的动力学。密克罗尼西亚联邦已经看到 在细胞骨架动力学研究中的广泛应用。在世界银行的赞助下 目前，我的实验室已经开发出使FSM成为 定量成像技术(QFSM)。与几个实验小组合作，如 通过在我自己的实验室中开发FSM成像功能，我们使用了QFSM技术 肌动蛋白和微管细胞骨架及其相关分子的动力学研究 结构在细胞形态发生、迁移和分裂中的作用；并将该方法扩展到 细胞信号转导中细胞表面受体的瞬时组装分析。由于它的刚性 然而，对衍射受限成像QFSM的要求仅限于实时成像 在玻璃片上培养的细胞，这是完全不生理的。利用最近的 光片成像的革命我们在这里建议将QFSM有序地带到第三维 将其力量应用于揭示细胞和细胞的器官型模型中的细胞骨架动力学 纸巾。这项工作将需要i)设计和实现的迭代优化 多光谱光片显微镜；ii)灵活而同时的亚化学计量比 多个大分子组装体的标记，III)计算工具的发展 三维时移体散斑动力学的跟踪与解释。具体来说，在目标1中， 我们将专注于追踪微管和末端的分子聚集体以及笼状蛋白涂层。 Pits将开发快速3D图像采集和高灵敏度3D粒子跟踪方法 测量大分子组件寿命的目标。在目标2中，我们将重点介绍 细胞极化过程中肌动球蛋白细胞皮质的动态变化以形成强健的双色 基于散斑产生和光流的计算方法，目标是 细胞皮质中分子周转和收缩的时空标记率 网络。在目标3中，我们将重点研究肌动蛋白细胞皮质之间的相互作用， 细胞黏附，以及细胞的胶原性3D微环境，以同时发育4- 散斑图案的彩色图像采集及其量化和计算工具 可视化大分子组装的耦合动力学，目的是测试 3D中，皮质网络和细胞基质粘连之间存在分子离合器的假设为零。 所有工具的设计都将着眼于推广，以便我们的技术 创新可以快速部署到社区，用于研究其他动态细胞 结构。

ABSTRACT Fluorescent Speckle Microscopy (FSM) is an imaging mode to visualize and quantify the dynamics of macromolecular assemblies in living cells. It relies on vastly substoichiometric labeling of one or several components of the assembly of interest. When imaged by diffraction limited optics this labeling generates a random punctate texture that encodes in a statistical fashion transport, mechanical deformation, and molecular turnover of the assembly. As such, FSM is related to the super-resolution techniques STORM and PALM, which both rely also on random sampling of the molecular constituents of macromolecular assemblies. In STORM and PALM substochiometry in labeling is achieved by passive or active switching of a small set of fluorescent probes between a dark and a bright state. In contrast, in the original implementation FSM has relied on a population of permanently labeled subunits that dynamically incorporate in the assembly. In STORM and PALM, substochiometric labeling is exploited to sequentially collect the coordinates of individual subunits with nanometer precision, i.e. to acquire over time a super-resolution map of the molecular organization of an assembly. In FSM, substochiometric labeling is exploited to track in real-time subunit motion, addition and removal. Accordingly, the spatial resolution of FSM is still diffraction-limited; however, FSM offers information about the dynamics of a macromolecular assembly no other imaging modality provides. FSM has seen widespread applications in the research of cytoskeleton dynamics. Under the auspices of the present grant, my lab has developed the computational approaches required to make FSM a quantitative imaging technique (qFSM). In collaboration with several experimental groups as well as by developing FSM imaging capabilities in my own lab we have used qFSM technology to study the dynamics of the actin and microtubule cytoskeletons and associated molecular structures in cell morphogenesis, migration and division; and extended the method to the analysis of transient assembly of cell surface receptors in cellular signaling. Due to its rigid requirements for diffraction-limited imaging qFSM has been restricted, however, to live imaging of cells cultured on glass slides, which is entirely unphysiological. Capitalizing on the recent revolution in light-sheet imaging we propose here to take qFSM to the third dimension in order to apply its power for unveiling cytoskeleton dynamics in organotypic models of cells and tissues. This endeavor will require an iterative optimization of i) the design and implementation of multispectral light-sheet microscopy; ii) the flexible and simultaneous, substoichiometric labeling of multiple macromolecular assemblies, iii) the development of computational tools for tracking and interpretation of speckle dynamics in 3D time-lapse volumes. Specifically, in Aim 1, we will focus on molecular aggregates tracking microtubule plus ends and on clathrin-coated pits to develop fast 3D image acquisition and highly-sensitive 3D particle tracking methods with the goal of measuring the lifetime of macromolecular assemblies. In Aim 2, we will focus on the dynamics of the actomyosin cell cortex during cell polarization to develop robust dual-color speckle generation and optical-flow based computational methods with the goal of spatiotemporally mapping rates of molecular turnover and contraction in the cell cortical network. In Aim 3, we will focus on interactions between actomyosin cell cortex, components of cell adhesions, and the collagenous 3D microenvironment of cells to develop simultaneous 4- color image acquisition of speckle patterns and the computational tools for quantification and visualization of the coupled dynamics of macromolecular assemblies with the goal of testing the null hypothesis of a molecular clutch between cortical network and cell matrix adhesions in 3D. All tools will be engineered with an eye towards generalization, so that our technological innovations can be rapidly deployed to the community for the study of other dynamic cell structures.