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）： 我们开发了一种强大的新型荧光散斑显微镜 (FSM) 方法，利用落射荧光显微镜来分析体内和体外大分子结构的运动、组装和分解。最初，我们设计这种方法是为了研究细胞骨架聚合物、微管和肌动蛋白丝在细胞运动和分裂中的动力学。在 FSM 中，结构由少量荧光标记的亚基和未标记的亚基组装而成，并使用高分辨率光学器件和灵敏的低噪声数码相机进行成像。 FSM 是通过显微注射或表达少量荧光团共轭亚基在活细胞中实现的。每个分辨率有限的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，散斑强度的运动和变化充当结构易位、组装和拆卸的报告者。 FSM 正迅速成为细胞骨架动力学研究的首选方法。然而，FSM 需要进一步发展才能充分发挥其生物医学研究的潜力。成像过程中的焦点漂移是 FSM 伪影的主要来源，必须证明 FSM 对于各种荧光显微镜模式的多功能性。此外，延时FSM图像的分析目前都是手工完成的，速度慢、不完整且容易出错。这必须被自动化、定量的 FSM 图像分析所取代。最后，FSM 图像生成和分析技术必须扩展到一般生物大分子组装体。为了实现这些目标，我们有以下具体目标： 1. 硬件开发：开发焦点稳定的 FSM 和多光谱全内反射 FSM (TIR-FSM)，用于体外研究蛋白质动力学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之外的其他生物学重要系统。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。