Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC)

作为对NIH RFA-RM-19-009的回应，我们建议建立Stanford-SLAC CryoET标本 筹建服务中心要实现四个具体目标：（1）搭建平台， 对于整个科学界来说，简化适合下游低温的样品的制备 电子断层扫描（cryoET）数据收集;（2）提供先进的冷冻标本制备 广泛的样品技术：大分子复合物，生物材料的微晶，细胞 裂解物、细胞器、细胞和组织;（3）为新用户编写关于制备 冷冻、水合生物样本，可选择进行相关冷冻荧光 冷冻聚焦离子束扫描电子显微镜（cryoFIB）和冷冻ET;以及（4） 采用新的样品制备方法，使用其他地方开发的创新技术。在这一提议中， 我们将利用我们现有的设施和内部专业知识，在以下每一种成像模式及其 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 培训上述议定书新用户的课程材料。我们将调整现有的基础设施 管理项目行政和资源分配。提供管理支持，以帮助用户 住宿安排，以及实验室入职和标本生物安全批准。我们有一个很好的集合- 为远程用户提供通信基础设施，以参与实际实验，特别是cryoFLM， cryoFIB和cryoET。我们将提供现有的Talos Arctica和/或Titan Krios显微镜，以评估 准备好的样品是否已准备好在相关的中心进行数据收集，该中心将在另一个 独立的NIH支持机构。我们将把现有的斯坦福-SLAC冷冻标本制备 在本提案获得资金后，立即提供有限的设备。我们预计将服务约40名用户 一旦中心在奖励期的第二年开始全面运作，每年支付一次。我们也会提供普通人手- 每年培训约10-12名新用户的讲习班。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。用户数量将在 在随后的几年里，我们和我们的经常性用户优化了中心资源的使用。在第3-6年，我们 将与合作者密切合作，开创先进的协议，如半自动化和自动化 cryoFIB和组织样本的“提升”，以便在拟议的中心实施。最后，我们将组装一个 科学的专家咨询委员会，指导我们的服务中心的做法和正在进行的 实施尖端技术。

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.