Quantitative Fluorescent Speckle Microscopy

定量荧光散斑显微镜

• 批准号: 6991224
• 负责人: Clare Michal Waterman
• 金额: $ 40.63万
• 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 2007-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6991224
• 关键词: Quantitative; Fluorescent; Speckle; Microscopy

DESCRIPTION (provided by applicant): We have developed a powerful new Fluorescent Speckle Microscopy (FSM) method that utilizes epi-fluorescence microscopy to analyze the movement, assembly, and disassembly of macromolecular structures in vivo and in vitro. Initially, we devised this method for studying the dynamics of the cytoskeletal polymers, microtubules and actin filaments in cell locomotion and division. In FSM, structures are assembled from a low fraction of fluorescently labeled subunits together with unlabeled subunits and imaged with high-resolution optics and a sensitive, low-noise digital camera. FSM is achieved in living cells by microinjection or expression of a low amount of fluorophore-conjugated subunits. Stochastic variations in the number of fluorescent subunits per resolution-limited image region results in a "speckled" appearance of the assembled structure. In time-lapse FSM, movement and changes in speckle intensity act as reporters for structure translocation, assembly and disassembly. FSM is fast becoming the method of choice for studies of cytoskeleton dynamics. However, FSM requires further development to reach its full potential for biomedical research. Focus drifts during imaging are a major source of FSM artifact, and the versatility of FSM for various fluorescence microscope modes has to be demonstrated. In addition, the analysis of time-lapse FSM images are currently done by hand, which is slow, incomplete, and prone to error. This must be replaced by automated, quantitative FSM image analysis. Finally, FSM image generation and analysis techniques have to be broadened to general biomacromolecular assemblies. To achieve these goals, we have the following Specific Aims: 1. Hardware Development: To develop focus-stabilized FSM and multi-spectral Total Internal Reflection FSM (TIR-FSM) for studying protein dynamics in vitro and on ventral cell surfaces in vivo. 2. Software Development: To develop software for automatic quantitative analysis of protein dynamics in time-lapse FSM images. 3. Applications: To use multi-spectral FSM, TIR-FSM, and analysis software for studying other biologically important systems besides cytoskeletal polymers. These advances will allow FSM to become a powerful tool for studying molecular dynamics in cell biology, neurobiology, biotechnology, and material science.

描述（由申请人提供）： 我们已经开发了一种强大的新荧光斑点显微镜（FSM）方法，该方法利用了表观荧光显微镜来分析体内和体外大分子结构的运动，组装和拆卸。最初，我们设计了这种方法来研究细胞运动和分裂中细胞骨架聚合物，微管和肌动蛋白丝的动力学。在FSM中，结构是从荧光标记的亚基和未标记亚基的低比例组装的，并使用高分辨率光学器件和敏感的低噪声数码相机成像。通过微注射或低量荧光团偶联亚基的表达来实现FSM。每个分辨率受限图像区域的荧光亚基数量的随机变化导致组装结构的“斑点”外观。在延时FSM中，运动强度的运动和变化是结构转运，组装和拆卸的记者。 FSM迅速成为细胞骨架动力学研究的首选方法。但是，FSM需要进一步的发展才能实现其生物医学研究的全部潜力。成像过程中的焦点漂移是FSM伪影的主要来源，并且必须证明FSM在各种荧光显微镜模式中的多功能性。此外，目前手工完成了延时FSM图像的分析，这是缓慢，不完整且容易出错的。必须由自动化的定量FSM图像分析代替。最后，必须将FSM图像的产生和分析技术扩展到一般的生物ac骨分子组件。为了实现这些目标，我们具有以下具体目标：1。硬件开发：开发稳定的FSM和多光谱的总内反射FSM（TIR-FSM），用于研究体外和体内腹侧细胞表面的蛋白质动力学。 2。软件开发：开发用于延时FSM图像中蛋白质动力学自动定量分析的软件。 3。应用：使用多光谱FSM，TIR-FSM和分析软件来研究除细胞骨架聚合物以外的其他重要系统。这些进步将使FSM成为研究细胞生物学，神经生物学，生物技术和材料科学中分子动力学的强大工具。