Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC)

斯坦福-SLAC CryoET 标本制备服务中心 (SCSC)

基本信息

批准号：
10054621
负责人：
Wah Chiu
金额：
$ 168.9万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10054621
关键词：
Academia Adopted Advisory Committees Area Award Biocompatible Materials Biological Biomedical Research Cells Cellular Structures Collaborations Communication Communities Complex Cryo-electron tomography Cryoelectron Microscopy Data Collection Educational Curriculum Educational workshop Equipment Evaluation Fluorescence Microscopy Freezing Funding Goals Grant Hydration status In Situ Industry Infrastructure Institution Investigation Ions Laboratories Lifting Macromolecular Complexes Methods Microscope Molecular Structure Organelles Preparation Process Professional Organizations Protocols documentation Request for Applications Research Research Personnel Resolution Resource Allocation Resources Sampling Scanning Electron Microscopy Schedule Scientist Selection Criteria Services Specimen Subcellular structure Techniques Technology Tissue Sample Tissues Titan Training Training Activity United States National Institutes of Health Work cryogenics data management electron tomography experience experimental study imaging modality innovative technologies instrument instrumentation interest member operation programs response structural biology symposium tool web portal

项目摘要

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.

为了响应 NIH RFA-RM-19-009，我们建议建立斯坦福-SLAC CryoET 样本准备服务中心（SCSC）要完成四个具体目标：（1）建立一个可用的平台对整个科学界来说，简化适合下游低温的样品制备电子断层扫描 (cryoET) 数据收集；(2) 提供先进的冷冻标本制备方法适用于多种样品的技术：大分子复合物、生物材料微晶、细胞 (3) 为新用户制定培训课程冷冻、水合的生物样本，可选择进行相关冷冻荧光光分析显微镜 (cryoFLM)、冷冻聚焦离子束扫描电子显微镜 (cryoFIB) 和 CryoET；以及 (4) 在本提案中，采用使用其他地方开发的创新技术进行样品制备的新方法。我们将利用我们现有的设施和内部专业知识来处理以下每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 我们将建立所有这些。我们将准备视频，用于拟议的服务和培训活动。培训新用户使用上述协议的课程材料我们将调整我们现有的基础设施。管理项目管理和资源分配管理支持已到位，以帮助用户萎缩。住宿安排以及实验室入住和样本生物安全批准我们有一套完善的安排。为远程用户参与实际实验（尤其是 Cryoflm）提供通信基础设施，我们将提供现有的 TALOS ARCTICA 和/或 TITAN KRIOS 显微镜进行评估。是否为关联的集线器中的数据颜色创建准备好样本，这将在另一个集线器中设置我们将在独立的 Nih 支持下进行现有的 SLAC 冷冻标本制备。本提案资助后，我们预计将立即为约 40 名用户提供有限的设备。在奖励期的第 2 年初，中心的每年是完全可选的。在研讨会培训中 ~ 每 10-12 个新用户我们将向广大社区展示我们的资源。通过门户网站和专业协会年会的展位，用户数量将会增加。在接下来的几年中，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。将与开创先进协议（例如半自动和自动化）的合作者密切合作冷冻聚焦离子束（cryoFIB）和组织样本的“提取”以在拟议的中心实施。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实践和正在进行的植入前沿技术。