Conventional bright-field electron tomographic tilt series are obtained by collecting electrons that have traversed a specimen illuminated by a broad beam. Using this approach, the thickness is limited by the severe image blurring that occurs when electrons that have undergone multiple energy losses are focused by the objective lens of the microscope. Furthermore, the maximum area of the image is limited by the depth-of-field of the objective lens, so that only part of the sample is in focus at high tilt angles. Tomographic reconstruction using STEM with a tightly focused electron probe can overcome some of the limitations imposed by tomographic reconstruction using conventional TEM. First, because the incident STEM probe can be focused at any point in a specimen, large areas are imaged in focus even for high tilt angles. Second, because in STEM there are no image-forming lenses after the specimen, the resolution attainable in images of thick specimens is not further degraded by electrons that have suffered multiple energy losses. The most commonly applied STEM approach makes use of an annular dark-field detector to collect electrons that are scattered to high angles. However, the dark-field STEM technique is not well-suited to imaging thick biological specimens because of the limited depth of field defined by the large convergence angle of the incident electron probe. A tenfold or higher increase in depth of field is possible by adjusting the microscope optics to decrease the convergence semi-angle to approximately 1 mrad. Another limiting feature of dark-field STEM as applied to imaging thick specimens is the severe degradation in spatial resolution that occurs toward the bottom surface of a section because of beam broadening. In contrast, we found that much higher spatial resolution can be obtained by collecting only those electrons that are scattered to low angles, that is, by using an axial bright-field detector. We have applied STEM tomography to determine the 3D arrangement of the complex membrane systems that are present in blood platelets. Platelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their alpha-granules. Our aim was to address how the structural organization of alpha-granules supports their dynamics. We prepared platelets by high-pressure freezing and freeze-substitution to preserve the native state, and imaged nearly entire cells by recording tomograms in the scanning transmission electron microscope (STEM). In resting platelets, we observed a morphologically homogeneous alpha-granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations (i.e., macro-homogeneity). In resting platelets, the incidence of tubular granule extensions was low, 4%, but this increased by > 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially de-condensing alpha-granules and the canalicular system remained as separate membrane domains. De-condensing alpha-granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased about threefold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet alpha granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for the timed release of alpha-granule proteins. We have also applied the STEM tomography to visualize synaptic spines in cultured slices of rat hippocampus. It has been possible for the first time to visualize entire post-synaptic densities and to assess differences in ultrastructure that occur when certain important proteins such as PSD-95 are knocked down. In other experiments, it has been feasible to characterize entire ribbon synapses in rat retina and to visualize the precise organization of secretory vesicles within those structures. In another application of STEM tomography, we have analyzed presynaptic synaptic ribbons found at many synapses that convey graded, analog sensory signals in the visual and other sensory pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. We have used axial bright-field STEM tomography to obtain 3D structures of rat rod bipolar cell terminals in 1-micrometer-thick sections of retinal tissue at an isotropic spatial resolution of approximately 3 nm. The resulting structures could then be incorporated into numerical simulations of dynamical models to synaptic transmission. In another application, we have investigated the association of membranes with the mother centriole in the early stages of generation of primary cilia, which play essential roles in signal transduction. Defects in cilium formation or function cause ciliopathies. The advantage of STEM tomography in this study is its ability to provide 3D reconstructions of the entire centriole assembly. Taken together our work has demonstrated the feasibility and advantages of axial STEM tomography for imaging thick sections at a spatial resolution of 5 to 10 nm, which is comparable to the spatial resolution of conventional electron tomography from thinner sections (typically 3 to 8 nm). Most modern electron microscopes can be operated in STEM mode and can be readily equipped with a bright-field detector, which is anticipated to facilitate implementation of the technique. The demand for high-resolution, large-volume imaging of biological specimens has been addressed so far by the large-scale application of conventional electron tomography of thin sections. Our current work suggests that it will be possible to reconstruct intact organelles, intracellular pathogens and even entire mammalian cells through serial thick-section tomography. Correlative microscopy has become an essential technique for determining the relationship between structure and function in cells and tissues on the scale of subcellular organelles and supramolecular assemblies. We have used this approach to correlate images acquired in the transmission electron microscopy with x-ray fluorescence image acquired at the Advanced Photon Source, at Argonne National Laboratory. This workflow has allowed us to localize specific diffusible ions and hence help to elucidate the mechanism for uptake of manganese ions in Mn-enhanced magnetic resonance imaging of brain, a technique that enables the tracing of neuronal connections. Specimens of organotypic rat hippocampal slices were prepared by slam-freezing, and frozen cryosections were cryo-transferred into our TEM operating at a beam energy of 300 keV. Correlative images have provided evidence that divalent manganese ions are concentrated in Golgi vesicles surrounding cell nuclei, consistent with previously reported studies in which exocytosis of these vesicles provide a detoxification mechanism to remove the element from cell bodies. In other images it has been possible to correlate concentrations of manganese in synaptic structures within the hippocampal slices.
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