The Oxford Consortium for Three Dimensional Electron Microscopy

高分辨率细胞超微结构已经通过透射电子显微镜研究了40多年，这种方法彻底改变了我们对细胞器的形状和形式，细胞中的数量，它们如何分裂，单个细胞如何分裂和组织的理解。这些方法的最大问题之一是只能对非常薄的细胞切片进行成像，因此在没有复杂的连续切片和重建技术的情况下很难产生三维视图，并且也不可能产生定量数据。最近，透射电子断层摄影术已经成为从EM切片提取高分辨率三维信息的首选技术，并且对于细胞或组织的有限区域是非常强大的技术。该技术仍然是复杂的，虽然连续断层扫描可以观察整个细胞的三维结构，有严重的技术和时间限制，如果大面积的标本在相当深的研究需要在过去的十年中出现了两个新技术，我们相信是下一个革命性的变化，我们如何成像细胞和组织超微结构。这些是聚焦离子束铣削（FIB SEM），物理蚀刻掉样品的表面和连续块面显微镜（SBFM），通过从其表面去除薄切片来切割样品。这两种技术都使用扫描电子显微镜对嵌入样品的树脂块的表面进行成像。通过连续去除树脂切片，然后对树脂表面进行成像，收集一系列图像，这些图像可以重建成三维体积。牛津布鲁克斯大学和牛津大学威廉·邓恩爵士病理学院都拥有三维细胞电子断层扫描的专业知识，三名工作人员在美国博尔德的科罗拉多大学国际知名的三维成像中心接受过培训，每个PI都有关于三维成像的出版物。迄今为止，英国只有6台仪器，曼彻斯特（x2），莱斯特，伦敦大学学院，CR-UK伦敦研究所。一台致力于材料科学，5台致力于生物医学研究。提高这些技术的能力和人员培训，增加这些技术的优秀研究成果，应该被认为是英国生物科学研究的关键。我们的大部分应用数据都是在曼彻斯特进行的，那里对仪器的需求非常高。牛津地区没有这样的设施，也没有专门处理植物材料和向植物科学家提供标本制备建议的设施。有4个关键项目从PI的部门对这个补助金申请，我们将在未来三年内进行，其中几个的基础研究目前由BBSRC赠款支持。将立即开展一些具体项目，研究植物细胞内膜系统的结构、锥虫（昏睡病）寄生虫游泳器官的结构、核分裂机制和核内膜系统的发展。

High resolution cell ultrastructure has been studied by transmission electron microscopy for more than 40 years and this method has revolutionised our understanding of the shape and form of organelles, the number in a cell, how they divide, how individual cells divide and the organisation of tissue. One of the biggest problems with these methods are that only very thin slices of cells can be imaged so a three dimensional view was very difficult to produce without complex serial sectioning and reconstruction techniques and it is also impossible to produce quantitative data. More recently transmission electron tomography has become the technique of choice for extracting high resolution 3-dimensional information from EM sections and is a very powerful technique for limited areas of a cell or tissue. The technique is still complex, and although serial tomograms can be made to observe the 3-D structure of whole cells, there are severe technical and time limitations if large areas of specimen in considerable depth need to be studied.In the last decade two new techniques have emerged that we believe to be the next revolutionary change in how we image cell and tissue ultrastructure. These are focussed ion beam milling (FIBSEM), physically etching away the surface of a sample and serial block face microscopy (SBFM), cutting into the sample by removing thin sections from its face. Both techniques use a scanning electron microscope to image the surface of a resin block in which the sample is embedded. By continued removal of slices of the resin follwed by imaging the resin surface a series of images are collected that can be reconstructed into a three dimensional volume.In this proposal we will establish a three dimensional imaging consortium in the Thames Valley area. Oxford Brookes University and Sir William Dunn School of Pathology, University of Oxford both have expertise in three dimensional cellular electron tomography with three members of staff trained at the internationally renowned centre for three dimensional imaging at University of Colorado, Boulder, USA and each PI on the grant have publications on three dimensional imaging. To date there are only 6 instruments in the UK, Manchester (x2), Leicester, UCL, CR-UK London Res. Inst. One is dedicated to materials science and 5 to biomedical research. Increasing the capacity and training of staff in these techniques and increasing the output of excellent research in these techniques should be considered essential to UK bioscience research. Most of our applications data has been carried out in Manchester where demand on the instrument is extremely high. There is no such facility in the Oxford region, nor a facility with expertise to handle plant material and advise plant scientists on specimen preparation. There are 4 key projects from the PI's Departments on this grant application that we will undertake over the next three years and the underlying research for several of these is currently supported by BBSRC grants. Immediately a number of specific projects will be carried out on the structure of membrane systems within plant cells, the structure of the swimming apparatus of trypanosome (sleeping sickness) parasites, development of nuclear division mechanisms and membrane systems within the nucleus.