FLUORESCENT SPECKLE MICROSCOPY

荧光散斑显微镜

• 批准号: 7002662
• 负责人: EDWARD D. SALMON
• 金额: $ 46.27万
• 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 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7002662
• 关键词: Urodela; Xenopus; animal tissue; bioimaging /biomedical imaging; biomedical equipment development; centromere; computer program /software; computer system design /evaluation; fluorescence microscopy; fluorescent dye /probe; image enhancement; microtubules; mitotic spindle apparatus; small interfering RNA; technology /technique development; three dimensional imaging /topography; yeasts

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.

描述（由申请人提供）：荧光斑点显微镜（FSM）是我们最初开发的一种技术，用于测量有丝分裂期间纺锤体纤维内单个微管（MT）和肌动蛋白丝阵列的聚合/解聚运动和位点以及MT的极向通量。从含有低百分比的荧光标记的亚基（约1%或更少）的池中组装这些聚合物产生荧光团沿着聚合物晶格的随机分布，其产生在显微镜的衍射极限分辨率内从零到几个荧光团（5-8）变化的“荧光斑点”基准标记。本申请的主要重点是进一步发展有限状态机方法，用于主轴机械中MT功能的分析。特别是，MT和动粒蛋白如何在纺锤体组装，染色体对齐和准确的染色体分离中发挥作用。这需要开发新的FSM显微镜技术，用于快速记录MT荧光斑点相对于荧光标记或斑点在动粒，极点，MT相关蛋白（MAPs），马达蛋白和MT末端的多波长和3-D延时图像。FSM成为这些系统的强大分析工具的主要下一步是开发新的计算机视觉方法，用于以相对于纺锤体中的其他分子荧光标记的高分辨率获得关于聚合物在2-D和3-D中的运动和周转的定量信息。为了研究蛋白质的功能，我们特别感兴趣的是优化FSM的遗传模式生物，包括芽殖酵母，生物化学可访问的非洲爪蟾卵提取物和siRNA与哺乳动物组织细胞。在这些研究过程中获得的经验将用于指导和完善硬件和软件的开发。