CryoFIB Instrument for Biomedical CryoEM/ET Lamellae Preparation - Summary The California NanoSystems Institute (CNSI) at the University of California, Los Angeles (UCLA), is committed to pioneering nanobiology and biomedical research. Central to this mission, the Electron Imaging Center for Nanosystems (EICN), established in 2007, provides cutting-edge cryogenic electron microscopy (cryoEM) instruments and specialized expertise to elucidate the mechanisms of molecular machinery at the nanometer scale. A previous NIH S10 award was pivotal in installing the world’s first operational Titan Krios in 2008, a landmark achievement that fueled the cryoEM revolution of the past decade. This instrument produced data for one of the earliest near-atomic-resolution single-particle cryoEM structures and played a critical role in Moderna’s groundbreaking mRNA vaccine development. Building on this success, a second NIH S10 award enabled the acquisition of a Titan Krios G4 in 2024, equipped with advanced technologies for both cryoEM imaging and cryogenic electron tomography (cryoET) tilt series acquisition. These state-of-the-art instruments support hundreds of registered UCLA users, facilitating atomic-level structural analysis of isolated or enriched biological complexes for a wide range of research applications. While EICN’s cryoEM and cryoET capabilities meet current user needs, the lack of a cryogenic focused ion beam (cryoFIB) instrument significantly restricts our large user base from conducting in situ structural studies within cells and tissues using cryoET. Most cells, exceeding 1 µm in thickness, are too dense for electron penetration and require precise milling into lamellae of 200 nm or less to enable high-resolution cryoEM or cryoET imaging. This application seeks funding to acquire an Aquilos 2 cryoFIB instrument, which will empower NIH-supported biomedical research across 38 user laboratories (including 26 major users) within EICN’s extensive user community. The proposed cryoFIB will substantially enhance EICN’s cellular cryoET capabilities, advancing federally funded research in critical areas such as viral and microbial infections, neurodegenerative disorders, tumorigenesis, therapeutic development, drug design, and vaccine delivery systems, as well as the PI continuous cryoEM/cryoET method development. The Aquilos 2 cryoFIB will benefit from robust institutional support, including dedicated staff, ongoing instrument maintenance, and operational resources, ensuring long-term impact and accessibility. The instrument will be managed by EICN’s highly experienced core facility team, which has successfully operated the facility for 18 years since its inception. The principal investigator, managing director, facility technician, and user coordinator possess the technical expertise and operational experience necessary to ensure the cryoFIB’s seamless integration and productive use. This high-end S10 instrument will immediately bolster NIH- funded biomedical research across UCLA’s colleges of natural sciences, engineering, and the David Geffen School of Medicine, driving transformative discoveries in structural biology and beyond.
