We propose to add Three-Dimensional Electron Microscopy (3D EM) to our EM Shared Resource. We have provided microscopy service for 4 decades and are the longest continuously-running Shared Resource on campus. Many users need 3D reconstructions at the ultrastructural level. Currently, 3D analysis is attainable at Duke by confocal microscopy, but it gives only light microscopy resolution; using a commercial 3D EM laboratory is expensive. Addition of 3D EM capability would save grant funds and decrease turn-around time. With a Serial Block Face Scanning Electron Microscope (SBFSEM) large areas (cell-to-cell relationships), can be imaged at high resolution, automatically, and rapidly, with daa available in 1-2 days for reconstruction. Traditionally, ultrastructural 3D reconstruction has been done by serial ultrathin sectioning and finding the same area on many grids-a tedious and time-consuming job (months). Alternative procedures include Electron Tomography (ET) (but section thickness and amount of tilt are limited) and Focused Ion Beam (FIB) Microscopy (where area and depth are also limited), neither of which is available here. The Automated Tape-collecting UltraMicrotome (ATUM) has the advantage of preserving sections for reexamination, but requires keeping up with each of them and has issues of wrinkling and alignment in the SEM. Thus, we believe SBFSEM would best fit the varying needs of our investigators. SBFSEM is essential for all investigators herein. Two have already published 3D EM data; several have received 3D data on material we prepared and imaged at test facilities. Another, who spent months doing serial sectioning and routine transmission EM, has received SBFSEM data that we obtained. Two additional users have projects amenable to ET (unavailable here), but possible by SBFSEM; all others have confocal or MRI 3D experience and need 3D EM. All 17 projects are on identifying disease markers or treatment targets. Our proposal is viable. We have support of the Duke School of Medicine (Dean Nancy Andrews) and the Department of Pathology (Dr. David Howell) with operating funds and the Department of Pathology for space. We have ample data storage and analysis capability through the Core for In Vivo Microscopy (CVIM; Dr. Allan Johnson), the Light Microscopy Core Facility (LMCF; Dr. Sam Johnson), the CoreResearch@Duke (Dr. Iain Sanderson), the Duke Health Technology Systems (Dr. Billy Willis), and the Office of Duke Research Computing (Dr. Mark DeLong). We have a long history of service; the technical ability to prepare specimens, run the instrument, and collect and render the data sets to 3D (Dr. Mark Ellisman); and a 5-year Financial Plan showing that the proposal is sustainable. Please see our letters of support. Finally, for long-term stability, we have identified a younger colleague to assume the resource directorship after the next decade.
The Serial Block Face Scanning Electron Microscope would serve 17 different projects of at least 20 funded researchers, most of whom work on communication between cells and how metabolic processes are regulated. Most need large area (cell to cell, rather than intracellular) analyses so that serial block face sectioning is the method of choice, rather than Electron Microscopy Tomography or Focused Ion Beam Microscopy. Understanding normal pathways and comparing them to aberrant ones, examining interactions between different kinds of cells, studying cell organelles such as synapses, and elucidating mechanisms of infection or immunity will yield invaluable information on how to diagnose and treat diseases.
