Equipment for Correlative LM/EM by High Pressure Freezing and Tomography

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DESCRIPTION (provided by applicant): One of the most important developments in transmission electron microscopy in recent years has been instrumentation for automated electron tomography. Electron tomography (ET) produces 3D images of cell components at high resolution, helping bridge the gap between molecular structural biology and in vivo cell biological structure studies. In some tomography studies it is actually possible to recognize the signature structure of molecules within a 3-D model of the cell. Funds are requested for an Xplore 3D Retrofit package for a Tecnai 12 to upgrade an existing Tecnai 12 Biotwin electron microscope for automated tomographic data collection, and a Leica EMPACT2 high pressure freezer with Live Cell Imaging System to permit viewing of live cells in a light microscope, then to freeze them in about 5 seconds. This system of instruments will greatly enhance both the quality and quantity of electron tomographic data that can be obtained. Cells will be observed live in a light microscope and when they reach the right stage of the cell cycle or of development they will be rapidly frozen. After processing for electron microscopy, these same cells will be analyzed by ET and the structures of interest modeled and analyzed in 3D. This correlative light and electron microscopic approach will be an extremely efficient way to collect data. The data collection proposed in this application includes projects with direct health relevance. We will characterize the ultrastructure of a cell culture model system for studying breast cancer using these methods. We will analyze in high resolution, 3-dimensional detail how bacteria such as Mycobacterium tuberculosis infect mammalian cells, and we will study cell division in several cell types to understand how specific molecules can affect that process. The importance of cell division to research on cancer and abnormal development is well known. Each of these projects will benefit greatly by being able to film the phenomena of interest in live cells and rapidly transfer the specimen to the HPF for freeze fixation. Automated tomographic analysis will greatly improve the throughput of data for each project, making it possible to have statistically significant sample sizes. RELEVANCE. The goal of this research is to provide a high-resolution structural context for several disease-related processes in INTACT CELLS that correlates with the structural and functional information provided by molecular genetics and structural proteomics. The state-of-the-art equipment requested in this proposal will contribute significantly to our understanding of the mechanisms of bacterial infection, breast cancer, and how cells divide. In turn, this better understanding of mechanism will promote more effective treatments for disease intervention.