Quantitative Fluorescent Speckle Microscopy

超出所提供的空间。我们开发了一种强大的新型荧光斑点显微镜（FSM）方法，利用外显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，结构由一小部分荧光标记的亚基与未标记的亚基组合而成，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是在活细胞中通过微量注射或表达少量荧光团共轭亚单位实现的。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是结构移位、装配和拆卸的报告器。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，以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对各种荧光显微镜模式的通用性。此外，目前对延时FSM图像的分析是手工完成的，这是缓慢的，不完整的，并且容易出错。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的生物大分子组装。为实现这些目标，我们提出了以下具体目标：硬件开发：开发聚焦稳定FSM和多光谱全内反射FSM (TIR-FSM)，用于体外和体内腹侧细胞表面蛋白质动力学研究。2. 软件开发：开发软件，用于自动定量分析延时FSM图像中的蛋白质动力学。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外的其他重要生物学系统。这些进步将允许FSM成为一个强大的工具为研究分子动力学在细胞生物学、神经生物学、生物技术、和m_f_ri_lq q_i_n_网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. 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 m_f_ri_lq q_i_n_ PERFORMANCE SITE ========================================Section End===========================================