Acquisition of a Focused Ion Beam Scanning Electron Microscope with cryo-stage

Project Summary This proposal requests funds to acquire a Focused Ion Beam Scanning Electron Microscope (FIB-SEM) to support a diverse group of NIH-funded research projects at Vanderbilt University and Vanderbilt University Medical Center. This microscope will permit three-dimensional reconstruction of entire cell volumes with

sufficient spatial resolution to observe the fine details of organelles. This volume information is essential to understand and quantify the complex interactions of organelles and macromolecular complexes within cells. The addition of a cryo-stage will extend the functionality of the FIB-SEM, adding ion milling of vitrified frozen cells to

permit cryo-electron tomography of macromolecular structures in situ and by cryo-SEM. The Zeiss Crossbeam 550 is a state-of-the-art FIB-SEM with a high-performance ion column (3 nm resolution and up to 100 nA of probe current) that can efficiently perform FIB-SEM tomography and can be integrated with a Leica cryo-stage

to create thin lamella for cryo-electron tomography. Our major users will apply FIB-SEM tomography to break new ground on NIH-funded research projects which span a wide range of problems in cell, developmental, epithelial, intestinal, and cancer biology, among others. Projects include studies of cytoskeletal dynamics

(Tyska), assembly of cell surface features (Goldenring, Tyska), bacterial protein secretion (Lacy, Cover), basement membrane architecture (Weaver, Hudson), and cellular membrane trafficking (Weaver, Goldenring, Zhou). Together these investigations hold significance for our understanding of disease and illness related to

birth defects, cancer, digestive disorders, and many other human health problems. The Crossbeam 550 will be a collaboration between the Vanderbilt Cell Imaging Shared Resource (CISR) and the Vanderbilt Cryo-EM Facility (V-CEM) within the Center for Structural Biology (CSB). It will be administered by the Vanderbilt Cell

Imaging Shared Resource. The CISR will provide the infrastructure for introducing researchers across Vanderbilt to these new techniques, train users in operation of the instrument, and apply an established business model to support the long-term maintenance of the system. In summary, acquisition of the Zeiss Crossbeam 550 FIB-

SEM will allow for unprecedented volumetric imaging capabilities of biological samples at scales and spatial resolutions not currently available to the Vanderbilt community and will facilitate research on a diverse set of topics.