Quantitative Fluorescent Speckle Microscopy

定量荧光散斑显微镜

• 批准号: 8135496
• 负责人: Gaudenz Danuser
• 金额: $ 43.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 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 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8135496
• 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; 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; Phase; Polymers; Population; Property; Proteins; Regulation; Relative (related person); Resolution; Signal Transduction; Statistical Methods; Structure; System; Techniques; Technology; Testing; Variant; base; biological research; cell motility; fluorescence microscope; fluorophore; imaging modality; 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）：我们建议继续开发定量荧光散斑显微镜（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基的动态。在第一轮融资中，我们开发了焦点稳定的 TIRF FSM 和旋转盘共聚焦成像，用于组织培养细胞中的肌动蛋白细胞骨架和粘着斑动力学，并开发了图像分析软件，可将随机散斑图像信号转换为高分辨率的图谱。肌动蛋白丝的组装、拆卸和运输。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节，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新的范例。在这里，我们寻求迈出下一个关键步骤，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时空动力学和多个大分子结构相互作用的方法。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，这将使我们能够对活细胞中的肌动蛋白动力学进行原位生物化学分析。目标 2：将 qFSM 扩展到微管和中间丝细胞骨架的分析。目标 3：建立相关 qFSM 来探测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中关于细胞迁移中细胞骨架功能的基本机制的假设驱动的细胞生物学研究推动的，但我们的技术是普遍实施的，着眼于生物医学研究界的广泛使用。我们建议进一步开发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。我们的研究将集中于肌动蛋白丝、微管和中间丝细胞骨架系统动力学的空间和时间整合。这些相互作用集中涉及多种细胞功能。因此，所提出的进展将为细胞生物学界服务，以研究细胞生理学和病理行为的基本方面。