Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC)

项目摘要 为了响应NIH RFA-RM-19-009，我们建议建立斯坦福-SLAC CryoET标本制备服务中心（SCSC）将实现四个具体目标：（1） 建立一个供广大科学界使用的平台， 适合于下游低温电子断层摄影（cryoET）数据的样品的制备 收集;（2）提供先进的冷冻标本制备技术， 样品范围：高分子复合物，生物材料的微晶，细胞 裂解物，细胞器，细胞和组织;（3）为新用户提供关于 冷冻、水合生物标本的制备，可选择进行相关的 低温聚焦离子束扫描电子显微镜 （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 相关的样品制备工作流程：cryoFLM、cryoFIB和cryoET。我们将建立 所有必要的设备专用于拟议的服务和培训活动。我们将 制作录像教材，对新用户进行上述议定书方面的培训。我们将 调整我们现有的基础设施，以管理项目管理和资源分配。 行政支持到位，以帮助用户安排住宿，以及实验室 入职和样本生物安全批准。我们有一个良好的沟通机制 远程用户参与实际实验的基础设施，特别是cryoFLM， cryoFIB和cryoET。我们将提供现有的Titan Krios显微镜， 准备好的样品是否已准备好在相关中心进行数据收集， 在另一个机构设立，并得到NIH的单独支持。我们将把现有的斯坦福大学- 在此之后立即提供有限的SLAC冷冻标本制备设备 提案得到资助。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。 在奖励期的第二年开始运作。我们还将定期举办实践研讨会 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社会 通过门户网站和专业协会年度会议上的展位。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中心的使用， 资源在第3-6年，我们将与开拓先进协议的合作者密切合作 例如半自动化和自动化的冷冻FIB和用于组织样本的“提出”， 在拟议中的中心。最后，我们将组建一个科学咨询委员会， 专家指导我们的服务中心的做法和正在进行的实施切割- 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.