FLUORESCENT SPECKLE MICROSCOPY

描述(申请人提供)：荧光散斑显微镜(FSM)是我们最初开发的一种技术，用于在有丝分裂期间测量可移动组织培养细胞中单个微管(MTS)和肌动蛋白细丝阵列的聚合/解聚的运动和位置，以及纺锤体纤维内MTS的极向通量。从包含低百分比的荧光标记亚基(约1%或更少)的池中组装这些聚合物会产生沿聚合物晶格的荧光团的随机分布，从而在显微镜的衍射限制分辨率内产生从零到几个荧光团(5-8)的荧光斑点基准标记。这一应用的主要焦点是进一步发展用于主轴力学中MT函数分析的有限单元法。特别是，MT和动粒蛋白如何在纺锤体组装、染色体配对和准确的染色体分离中发挥作用。这就需要开发新的FSM显微镜技术，用于快速记录MT荧光斑点的多波长和3-D延时图像，这些斑点相对于运动中心、极点、MT相关蛋白(MAP)、马达蛋白和MT末端的荧光标记或斑点。FSM成为这些系统的强大分析工具的重要下一步是开发新的计算机视觉方法，以相对于纺锤体中的其他分子荧光标记获得关于聚合物在2-D和3-D中高分辨率运动和周转的定量信息。为了研究蛋白质功能，我们特别感兴趣的是优化遗传模式生物的有限状态机，包括发芽酵母，生物化学可获得的非洲爪哇卵提取物，以及哺乳动物组织细胞的siRNA。在这些研究过程中获得的经验将用于指导和改进硬件和软件的开发。

DESCRIPTION (provided by applicant): Fluorescent speckle microscopy (FSM) is a technique we initially developed for measuring the movements and sites of polymerization/depolymerization of individual microtubules (MTs) and arrays of actin filaments in motile tissue culture cells and the poleward flux of MTs within spindle fibers during mitosis. Assembly of these polymers from a pool containing a low percentage of fluorescently labeled subunits (about 1% or less) produces a random distribution of fluorophores along the polymer lattice that produces "fluorescent speckle" fiduciary marks varying from zero to several fluorophores (5-8) within the diffraction limited resolution of the microscope. The major focus of this application is on the further development of the FSM method for the analysis of MT function in spindle mechanics. In particular, how MT and kinetochore proteins function in spindle assembly, chromosome alignment and accurate chromosome segregation. This requires the development of new FSM microscope technology for the rapid recording of multi-wavelength and 3-D time-lapse images of MT fluorescent speckles relative to fluorescent marks or speckles at kinetochores, poles, MT associated proteins (MAPs), motor proteins and MT ends. A major next step for FSM to become a powerful analytical tool for these systems is the development of new Computer Vision methods for obtaining quantitative information about polymer movement and turnover in 2-D and 3-D at high resolution relative to the other molecular fluorescent markers in the spindle. To study protein function, we are particularly interested in optimizing FSM for genetic model organisms including budding yeast, for the biochemically accessible Xenopus egg extracts and for siRNA with mammalian tissue cells. Experience gained in the course of these studies will be used to direct and refine hardware and software development.