Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC)

为了响应NIH RFA-RM-19-009，我们建议建立Stanford-SLAC Cryoet标本 准备服务中心(SCSC)要完成四个具体目标：(1)建立一个可用的平台 对于广大科学界来说，简化了适合于下游低温的样品制备 电子断层扫描(CryoET)数据收集；(2)提供先进的低温样品制备 适用于多种样品的技术：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，可选择进行相关的低温荧光灯 显微镜(CryoFLM)、低温聚焦离子束扫描电子显微镜(CryoFIB)和低温电子显微镜；以及(4) 利用其他地方开发的创新技术，采用新的样品制备方法。在这份提案中， 我们将利用我们现有的设施和内部专业知识，在以下每一种成像方式及其 集成到相关的样品制备工作流程中：CryoFLM、CryoFIB和CryoET。我们将建立所有 专用于拟议服务和培训活动的必要设备。我们将准备视频 培训新用户了解上述协议的课程材料。我们将调整现有的基础设施 管理项目管理和资源分配。管理支持已到位，可帮助用户 住宿安排，以及实验室登船和标本生物安全审批。我们有一个很好的布景- 建立远程用户参与实际实验的通信基础设施，特别是对于CryoFLM， CryoFIB和CryoET。我们将提供对现有Talos Arctica和/或Titan Krios显微镜的访问，以评估 准备好的样本是否准备好在关联的中心进行数据收集，该中心将在另一个中心设置 有单独的NIH支持的机构。我们将把我们现有的斯坦福-SLAC冷冻标本准备工作 在这项提议获得资金后，立即提供有限的设备。我们预计将为约40名用户提供服务 每年一次，一旦中心在授权期的第二年初完全投入使用。我们还将提供定期人手- 关于研讨会，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的社区。 通过门户网站和专业协会年度会议的摊位。年，用户数量将增加 未来几年，随着我们和我们的经常性用户优化中心资源的使用。在3-6年级，我们 将与开创先进协议(如半自动和自动)的合作者密切合作 在拟建的中心，为组织样本实施低温离子交换膜和“拉出”技术。最后，我们将组装一个 科学咨询专家委员会指导我们服务中心的做法和正在进行的 实施前沿技术。

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 Talos Arctica and/or 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.