EM TOMOGRAPHY OF FAST-FROZEN, FREEZE-SUBSTITUTED CELLS

最近在电子显微镜、数字图像处理成像处理设备以及生物标本制备和分析方法方面的改进为细胞电子显微镜的革命奠定了基础。该计划项目汇集了科罗拉多大学的细胞生物学家，他们拥有不同的科学和技术经验，因此他们可以为此做出贡献，这将是重要的。我们的计划项目包括核心技术研究和开发的主要部分，其中我们将从快速冷冻的、冷冻替代的细胞中进行大量高效的EM断层扫描，其保存用于结构研究代表了最先进的技术。核心项目包括标记特定细胞大分子的新方法，结构建模技术的创新，标记特定细胞大分子的新方法，结构建模技术的创新，以及思考细胞器结构可变性的新方法。将有三个科学项目利用这项技术并促进其发展：分析哺乳动物细胞中的高尔基体结构和功能；描述酵母、藻类和植物中高尔基体的变异性和复杂性；对出芽酵母的中心体进行结构解剖；以及对哺乳动物动点的实验/描述性分析。这些项目中的每一个都将需要在核心开发的仪器和技术，但同时每个科学项目都将为开发的新技术的效力提供一个可行的试验台。每个项目的成员都习惯了合作努力，他们将这种改善细胞结构研究的综合方法视为未来5年研究的主要项目。

Recent improvements in electron microscopes in equipment for digital image processing imaging processing, and in methods for biological specimen preparation and analysis have set the stage for a revolution in cellular electron microscopy. This Program Project assembles cell biologists at the University of Colorado with diverse scientific and technological experience so they can make a contribution to this will be significant. Our program project includes a major section on Core Technological Research and Development in which we will implement highly efficient EM tomography of significant volumes from fast frozen, freeze-substituted cells whose preservation for structural study represents the state-of-the art. Core projects include novel approaches to the labeling of specific cellular macromolecules, innovations in structural modeling technology, and new approaches to the labeling of specific cellular macromolecules, innovations in structural modeling technology, and new approaches to thinking about the structural variability of organelles. Three scientific projects will capitalize on this technology and contribute to its development: an analysis of Golgi structure and function in mammalian cells; descriptions of Golgi variability and complexity in yeast, algae, and plants; a structural dissection of the centrosome from budding yeast; and an experimental/descriptive analysis of mammalian kinetochores. Each of these projects will require the instrumentation and technology developed in the Core, but at the same time each scientific project will provide a viable test bed for the efficacy of the new technologies developed. The members of each project are accustomed to collaborative effort, and they see this integrated approach to improving the study of cellular structure as a major project for their coming 5 years of research.