FLUORESCENT SPECKLE MICROSCOPY

荧光散斑显微镜

• 批准号: 6343110
• 负责人: EDWARD D. SALMON
• 金额: $ 18.71万
• 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/6343110
• 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.

荧光散斑显微镜（FSM）是我们发现的一种新技术，用于观察活细胞中大分子组装体的运动、组装和周转。当一小部分亚基具有荧光标记时，可以看到这些聚合物在其组装期间沿其晶格沿着获得荧光斑点分布。最高的散斑对比度是在约1%或更小的分数下实现的，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 μ m）的仅几个荧光团的可变数目。我们已经表明，包含1或2个荧光团的散斑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，需要进行许多改进，以使FSM更加强大并开辟新的应用程序。这些包括提高FSM的4-D（3D加时间）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图像测量散斑运动和寿命动力学的新方法，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）组装一个稳定的，精密的，多模式，多波长的数字显微镜和计算机系统的4-D的FSM; 2）使用在体外和活细胞模型的微管和肌动蛋白丝组装，以确定最佳分数的荧光标记的亚基的FSM使用荧光染料和GFP。应用产生每个亚基多个荧光团的方法，减少猝灭和光漂白的问题，并探讨其优点：3）从时间推移FSM图像堆栈的二维和三维轨迹和寿命;和4）在生物医学项目中测试新的FSM开发，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，以及测定微管旋转由微管马达蛋白所驱动。