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.
Statement We propose to establish the Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC) to meet the emerging national need for cryoET as a structural biology tool. The Center facility will enable external users to streamline the preparation of samples through providing access to, and support for, advanced cryo-specimen preparation techniques (cryoFLM, cryoFIB, and cryoEM/ET) for a wide range of samples with biomedical applications. The Center will offer a comprehensive program to train and cross-train scientists, regardless of their institutional affiliations, to become independent cryoET investigators.
