Quantitative Fluorescent Speckle Microscopy

定量荧光散斑显微镜

• 批准号: 7533130
• 负责人: Gaudenz Danuser
• 金额: $ 42.64万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7533130
• 关键词: Actins; Affect; Algorithms; Behavior; Biochemistry; Biomedical Research; Calibration; Cell Communication; Cell physiology; Cells; Cellular biology; Color; Communities; Complement; Computer Analysis; Computer software; Condition; Coupling; Cytoskeleton; Development; Drosophila genus; Epithelial Cells; Eye; Feedback; Filament; Fluorescence; Fluorescence Microscopy; Focal Adhesions; Funding; Goals; Image; Image Analysis; In Situ; Intermediate Filaments; Interphase; Kinetics; Label; Life; Maps; Measurement; Measures; Mediating; Methodology; Methods; Microfilaments; Microscopy; Microtubules; Molecular Structure; Morphogenesis; Motion; Movement; Neurons; Numbers; Phase; Polymers; Population; Property; Proteins; Quantitative Microscopy; Rate; Regulation; Relative (related person); Resolution; Signal Transduction; Statistical Methods; Structure; System; Techniques; Technology; Testing; Variant; base; biological research; cell motility; fluorescence microscope; fluorophore; interest; light microscopy; macromolecular assembly; molecular dynamics; monomer; novel; spatiotemporal; submicron; tissue/cell culture; tool

DESCRIPTION (provided by applicant): We propose to continue the development of quantitative Fluorescent Speckle Microscopy (qFSM). qFSM is a variant of fluorescence microscopy to analyze the dynamics of subunits within macromolecular structures in living cells. In the first round of funding, we developed focus-stabilized TIRF FSM and spinning disk confocal imaging of actin cytoskeleton and focal adhesion dynamics in tissue culture cells, and developed image analysis software that converts the stochastic speckle image signal into high- resolution maps of the assembly, disassembly, and transport of the actin filaments. We exploited the quantitative information delivered by this technology to study the regulation of the actin cytoskeleton in migrating epithelial cells and thus established a new paradigm for how the cell builds specific actin-based structures to drive directed cell migration. Here, we seek to make the next critical steps towards our long term goal of establishing qFSM as a method for the comprehensive analysis of spatiotemporal dynamics and interaction of multiple macromolecular structures in living cells. Our specific aims are: Aim 1: To extend qFSM to the measurement of absolute rates of polymer turnover which will allow us to perform in situ biochemistry of actin dynamics in living cells. Aim 2: To extend qFSM to the analysis of microtubule and intermediate filament cytoskeletons. Aim 3: To establish correlational qFSM to probe the dynamic interaction between two or more macromolecular structures in living cells. These developments are motivated by the hypothesis-driven cell biological research in our labs on the fundamental mechanisms of cytoskeletal function in cell migration, but our technology is implemented generically with an eye towards its broad use by the biomedical research community. We propose to further develop the technique of quantitative Fluorescent Speckle Microscopy for the analysis of the dynamics and interaction of macromolecular assemblies. Our studies will focus on the spatial and temporal integration of the dynamics of the actin filament, microtubule, and intermediate filament cytoskeleton systems. These interactions are centrally implicated in a wide array of cell functions. Thus, the proposed developments will serve the cell biology community to investigate fundamental aspects of cell physiology and pathological behaviors.

描述（由申请人提供）：我们建议继续发展定量荧光斑点显微镜（qFSM）。qFSM是荧光显微镜的一种变体，用于分析活细胞大分子结构内亚单位的动力学。在第一轮融资中，我们开发了聚焦稳定的TIRF FSM和旋转盘共聚焦成像肌动蛋白细胞骨架和组织培养细胞的焦点粘附动力学，并开发了图像分析软件，将随机散斑图像信号转换为肌动蛋白细丝的组装、拆卸和运输的高分辨率地图。我们利用该技术提供的定量信息来研究迁移上皮细胞中肌动蛋白细胞骨架的调控，从而为细胞如何构建特定的基于肌动蛋白的结构来驱动定向细胞迁移建立了一个新的范例。在这里，我们寻求下一步的关键步骤，以实现我们的长期目标，即建立qFSM作为一种综合分析活细胞中多个大分子结构的时空动态和相互作用的方法。我们的具体目标是：目标1：将qFSM扩展到聚合物周转绝对速率的测量，这将使我们能够在活细胞中执行肌动蛋白动力学的原位生物化学。目的2：将qFSM扩展到微管和中间丝细胞骨架的分析。目的3：建立相关的qFSM来探索活细胞中两个或多个大分子结构之间的动态相互作用。这些发展是由我们实验室对细胞迁移中细胞骨架功能基本机制的假设驱动的细胞生物学研究所推动的，但我们的技术是为了在生物医学研究界的广泛应用而普遍实施的。我们建议进一步发展定量荧光散斑显微镜技术，用于分析大分子组装体的动力学和相互作用。我们的研究将集中在肌动蛋白丝、微管和中间丝细胞骨架系统动力学的时空整合上。这些相互作用与一系列细胞功能密切相关。因此，提出的发展将服务于细胞生物学社区研究细胞生理和病理行为的基本方面。