Scanning Transmission Electron Tomography of Biological Structures

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

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 caused by chromatic aberration of the objective lens, which occurs when electrons undergoing multiple energy losses are focused into different planes by the objective of the microscope. Furthermore, the maximum area of the sample that can be imaged 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. Stain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including STEM tomography as well as serial blockface electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). We have developed a method to determine the stain density in conventionally prepared plastic sections, based on some straightforward measurements in the TEM. Numbers of stain atoms per unit volume are computed from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. We have used 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. We have also applied bright-field axial STEM tomography to deduce the structural arrangement of A-kinase anchoring protein (AKAP79/150), which helps organizes signaling proteins controlling synaptic plasticity in mammalian brain. AKAP79/150 associates with the plasma membrane and endosomes through its N-terminal domain that contains cysteine residues that are reversibly palmitoylated. Cysteine-to-serine mutations abolishing palmitoylation reduce its endosomal localization and association with the postsynaptic density (PSD). Thick-section STEM tomography revealed more AKAP immunogold-labeled clusters corresponding to endosomes in spines for wild-type AKAP than for cysteine-to-serine mutated AKAP, consistent with the requirement for AKAP palmitoylation in endosomal trafficking. Our data suggests that palmitoylation fine-tunes the nanoscale localization, mobility, and trafficking of AKAP79/150 in dendritic spines, which might have important effects on its regulation of synaptic plasticity (Chen et al., Frontiers in Synaptic Neuroscience, in press). We have 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 bright-field detectors, which is expected to facilitate implementation of the technique. Our current work shows that it is feasible to reconstruct 1- to 2-micrometer thick volumes of any tissue type that is prepared by fixation and embedding, and larger volumes can be reconstructed by serial thick-section STEM 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 provides 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.