This proposal requests funds to acquire a Cryo-Focused Ion Beam (Cryo-FIB) microscope system in support of a diverse group of NIH-funded investigators from four different departments at the Washington University School of Medicine. This microscope system will allow innovative nanomanipulation of vitrified cells and tissues into thin lamellae (~300 nm thickness) for Cryo-Electron Tomography (Cryo-ET), and will facilitate the in situ elucidation of three-dimensional structures of cellular organelles and macromolecular complexes at nanometer resolution. A key feature of this proposed platform is the high-degree of automation and integration with our existing Cryo-EM imaging workflow (Linkham Cryo stage for Light Microscopy, FEI Vitrobot, AutoGrid/cryo manipulation system, and FEI Titan Krios Cryo-TEM) that will enable the precise targeting of regions of interest within a sample. The proposed system will greatly impact the work of Six Major User Groups, which are focused on important biomedical questions regarding ciliopathic disease, neurodegenerative and neurological disorders, vascular malformation, regulation of hormone secretion, and toxic protein aggregation. These investigators all have established needs for the Cryo-FIB system to enable three-dimensional tomographic visualization of cellular macromolecules and organelles in situ in their most native state. Once established, we expect that this structural imaging capability will further support many other NIH-funded research programs at Washington University, fully leveraging our recent institutional investments in cellular imaging. The requested Scios 2 Cryo-FIB has been configured for a high degree of automation and integration with our existing Cryo-EM sample preparation and imaging workflow. The new instrument will be housed and maintained alongside this infrastructure in the Washington University Center for Cellular Imaging (WUCCI) to streamline operational efforts as well as maximize impact to all Washington University researchers. The space in which the proposed Cryo-FIB instrument would be housed is secure and easily accessible to all major users and is adjacent to both the WUCCI sample preparation laboratory (which contains various freezing apparatus including a Mark IV Vitrobot from FEI) as well as the Titan Krios Cryo-TEM imaging suite. The expertise and institutional support for this instrument are exceptional. Dr. James Fitzpatrick, the Scientific Director of WUCCI, and Dr. David Piston, the Chair of Cell Biology & Physiology and head of the WUCCI Advisory Board, are both world-renowned experts in cellular microscopy methods. The WUCCI is a recently created multi-disciplinary collaborative shared imaging technology resource center whose leadership brings over 30 years of combined experience in providing cost-efficient training and support for high quality quantitative cellular imaging to a wide range of NIH-funded users. To create the WUCCI, Washington University provided an initial investment of over $7M and dedicated over 6,500 square feet of specifically designed lab space. In addition, in 2016 Washington University committed another $7.2M of support to purchase the Titan Krios Cryo-TEM microscope.
Understanding the exact three-dimensional structural organization of proteins and macromolecular complexes in their native cellular environment is critically important to unraveling how changes in their behavior disrupt normal biological function and contribute to disease pathogenesis. Recent advances in Cryogenic Focused Ion Beam (FIB) milling now permit a complete Cryo-Electron Microscopy (Cryo-EM) imaging workflow from sample freezing, correlative cryo light microscopy, thin lamella (~300 nm) production, to Cryo-Electron Tomography. This workflow, which we term Cryo-FIB-Assisted Electron Tomography (Cryo-FAET), will enable in situ structural investigation of cellular organelles and macromolecular complexes involved in ciliopathic disease, neurodegeneration, cardiovascular disease, diabetes, and birth defects.