Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC)

项目摘要 为响应NIH RFA-RM-19-009，我们建议建立斯坦福-SLAC Cryoet样品准备服务中心(SCSC)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 适用于下游低温电子断层扫描(CREROET)数据的样品制备 收集；(2)提供获取先进的低温样品制备技术的广泛 样品范围：大分子络合物、生物材料微晶、细胞 裂解物、细胞器、细胞和组织；(3)为新用户编制关于 冷冻、水合生物标本的制备，可选择进行相关 低温荧光光学显微镜、低温聚焦离子束扫描电子 以及(4)采用新的样品制备方法。 其他地方开发的创新技术。在这项提案中，我们将利用我们现有的 以下每种成像模式及其集成方面的设施和内部专业知识 转化为相关的样品准备工作流程：CryoFLM、CryoFIB和CryoET。我们将建立 为拟议的服务和培训活动提供的所有必要设备。我们会 准备视频课程材料，对新用户进行上述协议培训。我们会 调整我们现有的基础设施，以管理项目管理和资源分配。 管理支持到位，帮助用户安排住宿，以及实验室 登机和标本生物安全审批。我们有一个很好的通讯系统 供远程用户参与实际实验的基础设施，特别是对于CryoFLM， CryoFIB和CryoET。我们将提供现有的泰坦·克里奥斯显微镜进行评估 准备好的样本是否准备好在关联的集线器中进行数据收集，这将 将在另一家机构设立，由NIH单独支持。我们将把我们现有的斯坦福大学- SLAC冷冻样品制备设备在此之后立即限量供应 提案得到了资金支持。我们预计，一旦中心完全建成，每年将为约40名用户提供服务 在授权期的第二年初开始运作。我们还将定期举办实践工作坊。 每年培训约10-12名新用户。我们将把我们的资源传播给广大的社区。 通过门户网站和专业协会年度会议的摊位。用户数量 在接下来的几年里，随着我们和我们的经常性用户优化使用该中心的 资源。在3-6年间，我们将与开创先进协议先河的合作者密切合作 例如半自动和自动CryoFIB和用于组织样本的“抬出”来实现 他们在拟建的中心。最后，我们将成立一个科学咨询委员会， 指导我们服务中心的实践和持续实施切割的专家- 边缘技术。

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.