Over the last few years we have continued development of ion-abrasion scanning electron microscopy which allows site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions over an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have extended the application of this method to a variety of eukaryotic cells and tissues to establish this as a powerful tool for cellular and sub-cellular imaging in 3D for biomedical and clinical applications. Highlights of progress over the last year include: (i) development of tools for correlative light and 3D electron microscopy, the first such demonstration showing that the same cells can be imaged first by fluorescence microscopy, and followed by imaging of the entire 3D volume by electron microscopy at resolutions that are higher by two orders of magnitude;(ii) quantitative analysis of mitochondrial structure in normal and diseased cells establishing new and fundamental principles relevant to their role in subcellular organization;(iii) development of a novel method called ?atom probe tomography? and demonstrating its first application to biology by imaging a HeLa cell at 1 nm resolution;(iv) development of new and improved methods for subvolume averaging in cryo-electron tomography leading to separation of closely related, yet distinct conformations of trimeric HIV and SIV envelope glycoproteins and (v) report of the first sub-nanometer resolution 3D structure of the HIV envelope glycoprotein, providing a structural platform for vaccine design.
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