NIH / OD – ORIP
CRYO-FIB-SEM SYSTEM FOR THE NANOSCALE IMAGING AND MANIPULATION OF FROZEN BIOLOGICAL SPECIMENS
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 Aquilos Cryo-FIB has been configured for a high degree of automation and integration with our existing Cryo-EM sample preparation and imaging workflow.