Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC)

斯坦福-SLAC CryoET 标本制备服务中心 (SCSC)

• 批准号: 10818212
• 负责人: Wah Chiu
• 金额: $ 330.19万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-16 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10818212
• 关键词: Academia; Adopted; Advisory Committees; Area; Award; Biocompatible Materials; Biological; Cells; Collaborations; Communication; Communities; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Data Collection; Dedications; Educational Curriculum; Educational workshop; Electron Microscope; Equipment; Fluorescence Microscopy; Freezing; Funding; Goals; Hydration status; Ice; In Situ; Industry; Infrastructure; Institution; Ions; Laboratories; Lifting; Macromolecular Complexes; Methods; Microscope; Organelles; Preparation; Process; Professional Organizations; Protocols documentation; Recurrence; Request for Applications; Research; Resource Allocation; Resources; Sampling; Scanning; Scanning Electron Microscopy; Services; Specimen; Subcellular structure; Techniques; Technology; Thick; Thinness; Tissue Sample; Tissues; Titan; Training; Training Activity; United States National Institutes of Health; Work; cryogenics; electron tomography; experimental study; imaging modality; innovative technologies; nanometer resolution; response; structural biology; symposium; tool; web portal

Project Summary In response to the NIH RFA-RM-19-009, we propose to establish the Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC) to accomplish four specific aims: (1) establish a platform, which is available to the scientific community at large, to streamline the preparation of samples suitable for downstream cryogenic electron tomography (cryoET) data collection; (2) provide access to advanced cryo-specimen preparation techniques for a wide range of samples: macromolecular complexes, microcrystals of biological materials, cell lysates, organelles, cells and tissues; (3) generate a training curriculum for new users on the preparation of frozen, hydrated biological specimens, with the option of carrying out correlative cryo-fluorescence light microscopy (cryoFLM), cryo-focused ion beam scanning electron microscopy (cryoFIB), and cryoET; and (4) adopt new methods for sample preparation using innovative technologies developed elsewhere. In this proposal, we will leverage our existing facilities and in-house expertise in each of the following imaging modalities and their integration into a correlated sample preparation workflow: cryoFLM, cryoFIB, and cryoET. We will establish all the necessary equipment dedicated to the proposed service and training activities. We will prepare video curriculum materials to train new users in the aforementioned protocols. We will adapt our existing infrastructure to manage project administration and resource allocations. Administrative support is in place to help users with lodging arrangements, as well as laboratory on-boarding and specimen biosafety approvals. We have a well set- up communication infrastructure for remote users to participate in actual experiments, particularly for cryoFLM, cryoFIB, and cryoET. We will provide access to existing Titan Krios microscopes to evaluate whether the prepared samples are ready for data collection in the associated Hub, which will be set up at another institution with separate NIH support. We will make our existing Stanford- SLAC cryo-specimen preparation equipment available on a limited basis immediately after this proposal is funded. We anticipate serving ~40 users per year once the Center is fully operational in early Year 2 of the award period. We will also offer regular hands- on workshops to train ~10-12 new users per year. We will disseminate our resources to the broad community via a web portal and booths at professional societies’ annual conferences. The number of users will increase in subsequent years as we and our recurring users optimize the use of the Center’s resources. In Years 3-6, we will work closely with collaborators pioneering advanced protocols such as semi-automated and automated cryoFIB and “lift-out” for tissue samples to implement them in the proposed Center. Lastly, we will assemble a Scientific Advisory Committee of experts to guide the practices of our Service Center and the ongoing implementation of cutting- edge technologies.

项目概要 为了响应 NIH RFA-RM-19-009，我们提议建立斯坦福-SLAC CryoET 标本制备服务中心 (SCSC) 旨在实现四个具体目标：(1) 建立一个可供广大科学界使用的平台，以简化 制备适合下游低温电子断层扫描 (cryoET) 数据的样品 收藏; (2) 为广泛的人群提供先进的冷冻标本制备技术 样品范围：大分子复合物、生物材料微晶、细胞 裂解物、细胞器、细胞和组织； (3) 为新用户制定培训课程 制备冷冻、水合的生物样本，可选择进行相关分析 冷冻荧光光学显微镜 (cryoFLM)、冷冻聚焦离子束扫描电子 显微镜（cryoFIB）和cryoET； (4) 采用新的样品制备方法 其他地方开发的创新技术。在本提案中，我们将利用我们现有的 以下每种成像模式及其集成的设施和内部专业知识 集成到相关的样品制备工作流程中：cryoFLM、cryoFIB 和 CryoET。我们将建立 专用于拟议服务和培训活动的所有必要设备。我们将 准备视频课程材料来培训上述协议的新用户。我们将 调整我们现有的基础设施来管理项目管理和资源分配。 行政支持已到位，可帮助用户安排住宿和实验室 入职和样本生物安全批准。我们有良好的沟通机制 供远程用户参与实际实验的基础设施，特别是冷冻FLM， 冷冻FIB 和冷冻ET。我们将提供现有 Titan Krios 显微镜的访问权限以进行评估 准备好的样本是否已准备好在关联的 Hub 中进行数据收集，这将 在另一家拥有 NIH 独立支持的机构设立。我们将使我们现有的斯坦福大学- 在此之后，SLAC 冷冻标本制备设备将立即限量供应 提案得到资助。一旦中心全面投入使用，我们预计每年为约 40 名用户提供服务 在奖励期的第二年初期开始运作。我们还将定期举办实践研讨会 每年培训约 10-12 个新用户。我们将向广大社区传播我们的资源 通过门户网站和专业协会年会的展位。用户数 随着我们和我们的经常性用户优化中心的使用，在接下来的几年中将会增加 资源。在第 3-6 年，我们将与开创先进协议的合作者密切合作 例如半自动和自动冷冻FIB以及组织样本的“提取” 他们在拟议的中心。最后，我们将组建一个科学咨询委员会 专家指导我们服务中心的实践以及持续实施的削减- 边缘技术。