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

为了可视化细胞内的细胞骨架和膜组织，冷冻电子显微镜（cryoEM）提供了最佳分辨率，用于可视化组成细胞超微结构的微小组分。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，而不会引入改变或产生伪影。CryoEM对于所有“高影响力”分子细胞生物学研究至关重要，包括西蒙弗雷泽大学（SFU）“细胞生物学，发育和疾病中心（C2 D2）”的约30个成员实验室进行的研究。目前，在“SFU先进功能材料研究所（4D实验室）”可以使用普通的电子显微镜，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，使这些电子显微镜更容易获得生命科学研究，我们要求资金，以获得一个高压冷冻系统的cryoEM组织/细胞样品制备。** 我们建议为我们的机构提供其第一个高压冷冻系统，以满足所有SFU研究人员的常规电子显微镜需求，同时也使尖端的cryoEM实验成为可能（例如FIB-SEM）。Leica EM ICE易于使用，维护人员少，并且可以灵活地进行酵母、细菌和后生动物细胞的cryoEM样品制备，以及“软物质”研究。由于它的实用性，有压倒性的支持购买这个系统;有许多认捐，除了NSERC RTI资金将是必要的购买系统;包括四个SFU部门（MBB，BISC，BPK，Chem），科学学院，和一些个人研究人员（包括健康科学学院的实验室科学家委员会）。该建议的系统还允许将来升级到基础系统，用于研究电刺激后一毫秒发生的神经元事件。** 所提出的显微镜系统的灵活性与SFU分子细胞生物学实验室的各种NSERC资助的研究活动相匹配。领导这项应用，Beh实验室将使用拟议的系统直接可视化细胞内膜之间的微小接触位点，以了解膜系链复合物的调节组装。10名共同申请人（T博士）Beischlag，N.布兰达，M. Brockman，S. Gorski，N. Harden，H.哈特角Krieger，M. Silverman，G. Tibbits和E. Verheyen）将把新设备应用于各种研究领域，包括神经细胞转运、病毒因子-宿主膜相互作用、细胞间粘附和癌症的细胞病因学。作为所有SFU用户的开放资源，训练有素的学生将受益于掌握这一技术先进的系统，以便未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.**