LEICA EM UC/6 CRYO-ULTRAMICROTOME AND ACCESSORIES

描述(由申请人提供)： 2003年1月，《科学》杂志将低温电子断层扫描(GET)列为2002年十大科学突破之一。由于样品制备、EM仪器、软件和硬件方面的改进，现在从嵌在玻璃冰中的样品中收集高分辨率断层扫描数据变得更加现实。加州大学伯克利分校、旧金山分校、加州大学戴维斯分校和斯坦福大学都有或将会有电子显微镜来进行GET。对许多生物学家来说，研究小而完整的细胞是一种令人兴奋的选择，但也是有限的。研究较大细胞和组织的玻璃体冰冻切片为大多数研究人员提供了更多的可能性。为了为GET生产玻璃体冷冻切割机，需要资金购买徕卡EM UC6冷冻超微切割机和必要的附件。除了GET的尖端应用，该设备还将允许我们切割用于EM免疫标记的Tokuyasu型冷冻切片，以及用于常规(即非断层扫描)冷冻成像的超薄玻璃体冷冻切片。所有这些方法中的一种或多种将用于线虫、酿酒酵母、结核分枝杆菌、哺乳动物胃腺、内耳毛细胞立体纤毛和肾足细胞的野生型和突变表型。所有用于玻璃体冷冻的材料都将通过高压冷冻制备。显微镜将被安置在加州大学伯克利分校的电子显微镜实验室，这是一个收费的核心设施，向所有校园用户以及来自加州大学戴维斯分校、加州大学圣克鲁斯分校和斯坦福大学等周边机构的研究人员开放。这些机构中还没有一家拥有玻璃体冷冻切片所需的高压冷冻机和冷冻切割机。该提案中的所有主要用户都是经验丰富的新兴市场用户，而且大多数都有NIH的资助。选定的核心用户加上新兴市场实验室主任将组成一个咨询委员会，监督该工具的使用并制定政策。在核心调查员使用后的剩余时间内，将鼓励区域调查员使用。加州大学伯克利分校将提供空间、工作人员和资金来维护设备。

DESCRIPTION (provided by applicant): In January 2003, the journal Science listed Cryo Electron Tomography (GET) as one of the 10 Scientific Breakthroughs of 2002. Thanks to improvements in specimen preparation, EM instrumentation, software and hardware it is now more realistically possible to collect high-resolution tomographic data from samples embedded in vitreous ice. There are, or will be, electron microscopes at DC Berkeley, DC San Francisco, UC Davis, and Stanford to perform GET. Work on small, whole cells is an exciting, though limited option for many biologists. Working with vitreous cryosections of larger cells and tissues offers more possibilities for most researchers. To produce vitreous cryosections for GET, funds are requested for a Leica EM UC6 cryo-ultramicrotome and essential accessories. In addition to the cutting edge application of GET, this equipment will allow us to cut Tokuyasu-type cryosections for EM immunolabeling, and ultrathin vitreous cryosections for conventional, i.e., non-tomographic cryo-imaging. One or more of all these approaches will be used on wild type and mutant phenotypes in C. elegans, S. cerevisiae, Mycobacterium tuberculosis, mammalian gastric glands, inner ear hair cell stereocilia and kidney podocytes. All material for vitreous cryosections will be prepared by high pressure freezing. The microtome will be housed in the Electron Microscopy Laboratory at the University of California, Berkeley, a fee-for-service core facility open to all campus users as well as researchers from surrounding institutions such as UC Davis, UC Santa Cruz and Stanford. None of these institutions yet has the combination of high-pressure freezer and cryo-microtome required for vitreous cryo-sectioning. All the major users in this proposal are experienced EM users and most have NIH funding. Selected core users plus the Director of the EM Lab will make up an Advisory Committee to oversee use of the instrument and set policy. Use by regional investigators will be encouraged in the time remaining after core investigator use. UC Berkeley will provide space, staff, and funds for maintaining the equipment.