Over the last few years we have continued development of the use of focused ion beams in biology for 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. While FIB-SEM is now becoming a powerful tool in the arsenal for 3D biological imaging using electron microscopy, important limitations remain in the use of this nascent technology. First, the speed of data collection is limited by the time required for sequential SEM image acquisition and ion beam milling: imaging entire cellular volumes at 3D resolutions of 10 nm can take many days for completion. Second, the resolution of imaging is generally anisotropic and while effective pixel sizes as low as 3 nm can be obtained in the xy plane, obtaining the same resolution reproducibly in the z-direction has proven problematic. Finally, a problem that plagues imaging techniques in general is the forced trade-off between resolution and size. Small areas can be viewed at the highest possible resolution afforded by the technique, but larger fields of view must either be imaged at low resolution, or at high resolution by inefficient procedures such as tiling. These limitations in turn result in significant challenges for applications requiring true correlative imaging, where nanoscale objects are located using fluorescence microscopy, and the ultrastructure of the same regions are then determined by 3D electron microscopy. In order to address these issues, we have used a new keyframe imaging strategy that enables point-and-click high resolution 3D ultrastructural imaging of local regions of interest (ROIs), while also obtaining lower resolution ultrastructural information of the entire field of view. The technical advances that enable this are the following: (i) we target high-resolution imaging to only those regions which are of interest within a given field of view, (ii) we speed up the rate of acquisition by milling and imaging simultaneously instead of consecutively, (iii) we correct for drift in 3D, allowing the recording of images that consistently achieve resolutions better than 10 nm in all three planes and (iv) we combine this with light microscopy to identify regions of interest within a given volume. We show that all of these goals can be achieved for imaging both bacterial and mammalian cells using a commercially available microscope and demonstrate that a small nanoscale object such as a 100 nm-sized HIV core can be localized and imaged at 3D resolutions better than 10 nm within the cytoplasm of a 40 micron-wide mammalian cell, achieving correlative imaging across a volume scale of 10**9 in a single automated experimental run. Several applications of these approaches to unravel biological mechanisms are underway, on topics ranging from T cell signaling to bacterial cell division. In one recent application, we have focused on skeletal muscle cellular differentiation, a process that is governed by a dynamic relationship between both genetic and structural features. In our study, we analyzed changes in C2C12, an immortalized murine myoblast cell line, to explore the mechanistic pathways and structural changes that occurred during post-mitotic terminal differentiation for skeletal muscle cells. We applied super-resolutionstructure illumination microscopy (SIM) and quick substitution high pressure freezing - focused ion beam scanning electron microscopy (HPF/ FIB-SEM) imaging to view subcellular structures, including the nuclei, mitochondria, and nuclear pore complexes and mitochondria of myoblasts and myotubes in high resolution 3D images. Automated segmentation allows a quantitative look at a number of features of these cells. Our studies show that progenitor skeletal muscle cells, myoblasts, elongate the nuclei during the differentiation process. The changes in nuclear morphology support the alignment and subsequent fusion of cells to form long striated myotubes. Although the nuclear length increases, the nucleus and nucleolus volume decreased at the later stage of the differentiation process. These morphological reductions suggest a down-regulation in the cell proliferative activity, typical of cells undergoing terminal differentiation and settling into cell growth arrest. The reduction in nucleolus volume also reduces the zones of exclusion, providing more physical space for the expression of myogenic regulatory factors, which is distributed throughout the nuclei. Increased level of myogenic regulatory factors expression induces terminal differentiation and myogenesis. Although the size and shape of the nucleus changed during the differentiation process, the architectural pattern of nuclear pore complexes remained evenly distributed across the nuclear membrane. The number of nuclear pore complexes over a the nuclear surface area also remained the same across multiple nuclei from both myoblasts and myotubes, suggesting a specific organizational scheme that regulates the proximity of the nuclear pore complexes with respect to one another. Interestingly, and in contrast to these findings, it has been reported that the sizes of the nuclei and nucleolus and the number of nuclear pore complex significantly increase in cancer cells. In another application, we explored the ultrastructure of neural stem cells and activated T cells at cell-cell junctions. Our studies revealed close contact between the two cell types, with localization of the Golgi apparatus, vesicles, and mitochondria of the T cell to the cell-cell interface. 3D visualization revealed a cleft line running along the center of the T cell nucleus, surrounded by two compact clusters of mitochondria on opposite sides. The cleft line was directly facing the contact region, with direct evidence for engulfment by the T cell membrane of an edge of the neural stem cell at the contact zone. These observations suggest that the contact zones include active processes that involve participation of cytoskeletal elements. In addition to studies of the structural aspects of the interaction of HIV with cellular receptors, we have also investigated the mechanisms underlying cell-cell spread of the virus. HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. Our studies have led to a new paradigm in the field of cell-cell virus transmission, providing evidence that membrane extensions originating from uninfected cells, either as membrane sheets or filopodial bridges play a central role in HIV transmission from infected to uninfected cells.