We are requesting a dual-beam scanning electron microscope (SEM) that will be added to the shared electron microscopy facility at the New York Structural Biology Center (NYSBC). NYSBC currently provides state-of- the-art instrumentation and associated technical support for structural analysis of macromolecules by transmission electron microscopy (TEM), NMR and X-ray diffraction. These facilities are shared by a group of ten New York academic institutions, most of which are in New York City: Albert Einstein College of Medicine, City University of New York, Columbia University, Memorial Sloan-Kettering Cancer Center, Mount Sinai School of Medicine, New York University, The Rockefeller University, Wadsworth Center of the Department of Health, Weill Medical College of Cornell University, and the State University of New York. The primary goal of adding a dual-beam SEM is to expand the range of projects that can be addressed at NYSBC and specifically to offer a higher resolution alternative to cell biologists and neurobiologists who typically rely on light microscopy for morphological evaluation of tissue. To achieve this goal, we will implement an automated slice- and-view imaging mode, whereby the surface of a resin-embedded block of tissue is alternately imaged and etched, to generate a sequential series of 2D images through the tissue. A 3D rendering of the cell contents at 5-40 nm resolution is then obtained by using dedicated software to align and stack these images. An additional goal is to use a scanning transmission (STEM) detector to analyze the mass distribution of macromolecular complexes, thus facilitating structure determination using TEM imaging and single particle analysis. Additionally, we will use the dual-beam SEM for two development projects at NYSBC;namely, the manufacture of phase plates for TEM imaging and the thinning of frozen-hydrated cell cultures in order to fabricate suitable samples for cryoelectron tomography. Thirteen individual user projects are described, representing specific biological applications that will benefit from this technology. These include four neurobiology projects, two projects addressing pathogenesis of bacteria and HIV, and studies of cellular structures involved in autophagy, endocytosis, LDL processing and cell-cell adhesion. In addition, ongoing TEM studies of macromolecular complexes responsible for DNA and RNA replication and nuclear transport are described, which would benefit from STEM analysis and phase plate development. These researchers come from six different New York institutions and represent a sampling of research activities that would result from implementation of dual-beam SEM at NYSBC. By adding the new technologies provided by dual-beam SEM those already supported at NYSBC, we will significantly enhance its standing as a hub of structural biology in the greater New York metropolitan area.
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