FLUORESCENT SPECKLE MICROSCOPY

荧光散斑显微镜

• 批准号: 6044467
• 负责人: EDWARD D. SALMON
• 金额: $ 47.53万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6044467
• 关键词: animal tissue; bioimaging /biomedical imaging; biomedical equipment development; centromere; fluorescence microscopy; fluorescent dye /probe; image enhancement; microtubules; technology /technique development

Fluorescence Speckle Microscopy (FSM) is a new technique we have discovered for visualizing movement, assembly, and turnover of macromolecular assemblies in living cells. When a small fraction of subunits have fluorescent label, these polymers are seen to acquire a fluorescent speckle distribution along their lattice during their assembly. The highest speckle contrast is achieved for fractions of about 1% or less where the fluorescence of each speckle corresponds to a variable number of only a few fluorophores per resolvable unit (about 0.25 um) in the microscope. We have shown that speckles containing 1 or 2 fluorophores can be detected and recorded using a wide-field fluorescence light microscope, and digital imaging with a low noise cooled CCD camera. We are at very early stages in the development of SM and there are numerous improvements needed to take FSM much more powerful and open up new applications. These include improving the sensitivity, stability and resolution in 4-D (3D plus time) of FSM, combining FSM with other modes of light microscopy, determining new ways of measuring the kinetics of speckle motions and life-times from FSM images, and testing the developments in important collaborative projects. To achieve these goals, we have the following Specific Aims: 1) Assemble a stable, precision, multi-mode, multi-wavelength digital microscope and computer system for 4-D for FSM; 2) Use in vitro and living cell models for microtubles and actin filament assembly to determine the optimal fraction of fluorescently labeled subunits for FSM using fluorescent dyes and GFP. Apply ways of produce multiple fluorophores per subunit that are reduced in the problems of quenching and photobleaching, and explore their advantages; 3) trajectories and lifetimes in 2D and 3D from stacks of time-lapse FSM images; and 4) Test new FSM developments in biomedical projects investigating the motility and dynamics of microtuble and actin filament arrays in mitotic and migrating cells as well as in assaying microtuble rotation by microtuble motor proteins.

荧光斑点显微镜（FSM）是我们发现的一种用于观察活细胞中大分子组合的运动、组装和周转的新技术。当一小部分亚基具有荧光标记时，这些聚合物在组装过程中沿其晶格获得荧光斑点分布。最高的散斑对比度达到约1%或更少的分数，其中每个散斑的荧光对应于显微镜中每个可分辨单位（约0.25微米）的几个荧光团的可变数量。我们已经证明含有1或2个荧光团的斑点可以使用宽视场荧光显微镜和低噪声冷却CCD相机的数字成像来检测和记录。我们还处于SM开发的早期阶段，要使FSM更加强大并开发新的应用程序，还需要进行大量的改进。其中包括提高FSM在4-D （3D +时间）中的灵敏度、稳定性和分辨率，将FSM与其他光学显微镜模式相结合，确定从FSM图像中测量斑点运动动力学和寿命的新方法，以及测试重要合作项目的发展。为了实现这些目标，我们有以下具体目标：1)为FSM组装一个稳定、精密、多模式、多波长的数字显微镜和4-D计算机系统；2)利用体外和活细胞模型进行微管和肌动蛋白丝组装，利用荧光染料和绿色荧光蛋白确定FSM荧光标记亚基的最佳比例。应用每个亚基产生多个荧光团的方法，减少了猝灭和光漂白的问题，并探索其优点；3)延时FSM图像的二维和三维轨迹和寿命；4)在生物医学项目中测试FSM的新发展，研究有丝分裂和迁移细胞中微管和肌动蛋白丝阵列的运动性和动力学，以及通过微管运动蛋白分析微管旋转。