FLUORESCENT SPECKLE MICROSCOPY

荧光散斑显微镜(FSM)是我们发现的一种新技术，用于可视化活细胞中大分子组装的运动、组装和周转。当一小部分亚基有荧光标记时，这些聚合物在组装过程中可以看到沿着它们的晶格获得荧光斑点分布。对于大约1%或更少的分数，获得最高的散斑对比度，其中每个散斑的荧光对应于显微镜中每个可分辨单位(约0.25um)仅有几个荧光团的可变数量。我们已经证明，包含一个或两个荧光团的散斑可以用广场荧光显微镜探测和记录，并用低噪声冷却的CCD相机进行数字成像。我们正处于SM开发的早期阶段，需要进行大量改进才能使FSM变得更加强大并打开新的应用程序。这些措施包括提高FSM的灵敏度、稳定性和4-D(3D+时间)分辨率，将FSM与其他光学显微镜模式相结合，确定从FSM图像测量散斑运动动力学和寿命的新方法，以及测试重要合作项目的进展。为了实现这些目标，我们有以下具体的目标：1)组装一个稳定、精确、多模式、多波长的数字显微镜和计算机系统，用于FSM的4-D；2)使用体外和活细胞模型进行微管和肌动蛋白细丝组装，以确定使用荧光染料和GFP的FSM的最佳荧光标记亚基的比例。应用减少了猝灭和光漂白问题的每个亚基产生多个荧光团的方法，并探讨其优势；3)从层叠的延时FSM图像中获得2D和3D的轨迹和寿命；以及4)测试FSM在生物医学项目中的新进展，该项目研究有丝分裂细胞和迁移细胞中微管和肌动蛋白细丝阵列的运动性和动力学，以及通过微管马达蛋白检测微管旋转。

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.