Scanning Transmission Electron Tomography of Biological Structures

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications, trials & patents

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 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. 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. The method has been tested on specimens of embedded blood platelets, brain tissue and liver tissue (1). We have applied STEM tomography to perform 3-D imaging of 3-D micrometer thick sections of CA1 regions of mouse hippocampus to determine changes in astrocyte morphology that are associated with circadian rhythm set by the suprachiasmatic nucleus suprachiasmatic nucleus (SCN) of the hypothalamus. It was found that pyramidal neurons change the surface expression of NMDA receptors, and astrocytes change their proximity to synapses. Specifically, fewer astrocytic processes were detected in the D-phase than in the L-phase with a 2-fold increase in the nearest neighbor distance between each post-synaptic density and the closest astrocyte process (J.P. McCauley et al., Cell Reports 33: 108255; 2021) In another application, we 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. 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. We have used STEM tomography to investigate the association of membranes with the mother centriole in the early stages of generation of primary cilia, as these 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 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.