High-pressure freezing unit for visualizing cellular ultrastructure by electron microscopy

为了显示细胞内的细胞骨架和膜的组织，低温电子显微镜(Cryo-Electronics显微镜)为显示组成细胞超微结构的微小成分提供了最好的分辨率。通过在极端压力下快速冷冻样品，高压冷冻旨在保存复杂的细胞结构，而不会引入变化或产生人工制品。对于所有“高影响力”的分子细胞生物学研究，包括由西蒙弗雷泽大学(SFU)“细胞生物学、发育和疾病中心(C2D2)”的约30个成员实验室进行的研究，低温EM是必不可少的。目前，“SFU先进功能材料研究所(4D实验室)”有常用的电子显微镜，但它们是针对材料科学中使用的样品进行配置和优化的。为了方便使用，并使这些电子显微镜更容易用于生命科学研究，我们请求获得资金，以获得用于低温EM组织/细胞样本制备的高压冷冻系统。*我们建议为我们研究所提供其第一台高压冷冻系统，以满足所有SFU研究人员的常规电子显微镜需求，并使尖端低温EM实验成为可能(例如FIB-SEM)。徕卡EM ICE易于使用，只需最少的维护支持人员，并且可以灵活地使用酵母菌、细菌和后生动物细胞制备低温EM样品，以及进行“软物质”研究。由于其实用性，购买这一系统获得了压倒性的支持；除了NSERC RTI资金外，还有许多认捐资金将是购买该系统所必需的；包括四个SFU部门(MBB、BISC、BPK、Chem)、科学系和一些个人研究人员(包括卫生科学学院的实验室科学家委员会)。这一提议的系统还允许将来对基础系统进行升级，以研究电刺激后一毫秒发生的神经元事件。*拟议显微镜系统的灵活性与SFU分子细胞生物学实验室由NSERC资助的各种研究活动相匹配。作为这一应用的领头羊，BeH实验室将使用所提出的系统来直接可视化细胞内膜之间的微小接触位置，以了解膜系留复合体的受调控组装。这10位共同申请者(T.Beischlag博士、N.Branda博士、M.Brockman博士、S.Gorski博士、N.Harden博士、H.Hutter博士、C.Krieger博士、M.Silverman博士、G.Tibbit博士和E.Verheyen博士)将把新设备应用于各种研究领域，包括神经细胞运输、病毒因子-宿主膜相互作用、细胞间黏附和癌症的细胞病因学。作为向所有SFU用户开放的资源，受过培训的学生将从掌握这一技术先进的系统中受益，以便将来在生物技术领域就业。**

To visualize cytoskeletal and membrane organization within cells, cryo-electron microscopy (cryoEM) provides the best resolution for visualizing the tiny components comprising the cellular ultrastructure. By snap freezing samples at extreme pressures, high-pressure freezing is designed to preserve complex cellular structures without introducing alterations or generating artifacts. CryoEM is essential for all "high-impact" molecular cell biology research, including that conducted by the ~30 member labs of the "Centre for Cell Biology, Development and Disease (C2D2)" at Simon Fraser University (SFU). At present, common use electron microscopes are available at the "SFU Advanced Functional Materials Research Institute (4D labs)," but they are configured and optimized for samples used in material sciences. To both facilitate use and make these electron microscopes more accessible for life science research, we request funds to acquire a high-pressure freezing system for cryoEM tissue/cell sample preparation. ******We propose to provide our institution with its first high-pressure freezing system to serve both the routine electron microscopy needs of all SFU researchers, but also to make cutting-edge cryoEM experiments possible (e.g. FIB-SEM). The Leica EM ICE offers ease of use, minimal support personnel for upkeep, and flexibility for cryoEM sample preparation with yeast, bacteria, and metazoan cells, as well as "soft matter" research. Because of its utility, there is overwhelming support for the acquisition of this system; there are many pledged contributions, which in addition to the NSERC RTI funds will be necessary to purchase the system; including four SFU Departments (MBB, BISC, BPK, Chem), the Faculty of Science, and a number of individual researchers (including the Lab Scientists' Committee at the Faculty of Health Science). This proposed system also permits future upgrades to the base system for investigating neuronal events that transpire a msec after electrical stimulation. ******The flexibility of the proposed microscope system matches the varied NSERC-funded research activities of the molecular cell biology labs at SFU. Leading this application, the Beh lab will use the proposed system to directly visualize the minuscule contact sites between intracellular membranes to understand the regulated assembly of membrane tethering complexes. The 10 co-applicants (Drs. T. Beischlag, N. Branda, M. Brockman, S. Gorski, N. Harden, H. Hutter, C. Krieger, M. Silverman, G. Tibbits, and E. Verheyen) will apply the new equipment to various research interests including nerve cell transport, viral factor-host membrane interactions, intercellular adhesion, and cellular etiologies of cancer. As an open resource to all SFU users, trained students will benefit from the mastery of this technologically advanced system for future employment in biotech.**