2293 lines
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<article data-history-node-id="141576" about="/labs-at-nibib/laboratory-cellular-imaging-and-macromolecular-biophysics-lcimb/cellular-and-supramolecular-structure-and-function/research-topics" class="nibib-site-page node--nibib-site-page node--type-nibib-site-page">
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<div class="field field--name-field-card-title field--type-string field--label-hidden field__item">Quantitative STEM Analysis of Supramolecular Assemblies</div>
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<div class="field__item"> <img loading="lazy" src="/sites/default/files/CSSF%20image%20quantitative%20STEM%20analysis.png" width="433" height="415" alt="Dark-field scanning transmission electron microscopy of unstained fibrils of the arctic mutation of Alzheimer’s amyloid beta peptide, with co-deposited tobacco mosaic virus as a mass calibration standard. The mass measurements showed that the fibrils contained two or four beta sheets " title="Dark-field scanning transmission electron microscopy of unstained fibrils of the arctic mutation of Alzheimer’s amyloid beta peptide, with co-deposited tobacco mosaic virus as a mass calibration standard. The mass measurements showed that the fibrils contained two or four beta sheets (N. Norlin et al., J. Struct. Biol. 2012; 180; 174-189)." typeof="foaf:Image">
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<p>The purpose of this research is to develop techniques for determining quantitative structural information from supramolecular assemblies using the technique of scanning transmission electron microscopy (STEM). In STEM a finely focused probe of high-energy electrons is scanned across a biological structure of interest and various signals are detected at each image pixel. For example, we are using the annular dark-field signal to determine the molecular masses of large protein assemblies, e.g., Alzheimer’s disease-related amyloid fibrils; such measurements provide information about arrangement of subunits.</p>
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<div class="field field--name-field-card-title field--type-string field--label-hidden field__item">Novel Approaches for Determining 3D Cellular Ultrastructure </div>
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<div class="field field--name-field-card-text field--type-text-long field--label-hidden field__item"><p>We are also developing techniques for determining subcellular structure based on axial bright-field STEM tomography by recording images from micrometer-thick sections of cells over a range of tilt angles. Using this approach, we have shown that it is possible to reconstruct much thicker volumes than can be achieved with conventional TEM electron tomography. We are currently applying STEM tomography to visualize entire synapses in the nervous system at a spatial resolution of a few nanometers. Of particular current interest is to determine the structure of postsynaptic densities in hippocampal neurons, as well as ribbon synapses in rod bipolar cells of retina. We are also using STEM tomography to study a number of biological systems outside the field of neurobiology, including the structural changes that occur on activation of human blood platelets.</p>
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<div class="field field--name-field-card-title field--type-string field--label-hidden field__item">Quantitative Nanoscale Analysis using Inelastic Electron Scattering</div>
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<div class="field__item"> <img loading="lazy" src="/sites/default/files/CSSF%20image%20quantitiative%20nanoscale%20inelastic%20electron%20scattering.png" width="304" height="257" alt="Energy-filtered transmission electron microscopy of unstained section of mouse pancreatic islet of Langerhans, showing sulfur (blue), phosphorus (red), and nitrogen (green); insulin granules in the beta cell contain high sulfur, whereas glucagon granules in alpha cell contain low sulfur " title="Energy-filtered transmission electron microscopy of unstained section of mouse pancreatic islet of Langerhans, showing sulfur (blue), phosphorus (red), and nitrogen (green); insulin granules in the beta cell contain high sulfur, whereas glucagon granules in alpha cell contain low sulfur (M.A. Aronova, R.D. Leapman, MRS Bulletin 2012; 37: 53-62)." typeof="foaf:Image">
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<p>In this research, we are developing techniques to map chemical elements contained in subcellular structures using (1) energy-filtered transmission electron microscopy (EFTEM), and (2) STEM coupled with electron energy loss spectroscopy (EELS). In both approaches, we acquire hyperspectral inelastic images carrying information from characteristic excitations of atomic core-shells in the specimen. Our aim is to extract quantitative composition and to achieve a sensitivity of a few atoms. Recent applications include imaging the iron cores of individual ferritin molecules in neurons and other cells, where iron plays an important physiological role. In another application, we are combining EFTEM with electron tomography to map DNA and protein in three-dimensions within cell nuclei by using the phosphorus and nitrogen signals.</p>
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<div class="field field--name-field-card-title field--type-string field--label-hidden field__item">Methods for 3D Reconstruction </div>
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<div class="field__item"> <img loading="lazy" src="/sites/default/files/CSSF%20image%203D%20ultrastructure.png" width="386" height="398" alt="This is an image of a rendered three dimensional model of a malaria infected erythrocyte obtained by scanning transmission electron tomography" title="Rendered 3D model of a malaria-infected erythrocyte obtained by scanning transmission electron tomography (M.F. Hohmann-Marriott et al., Nature Methods 2009; 6: 729-732)." typeof="foaf:Image">
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<p>Tomographic reconstructions based on the standard weighted back-projection (WBP) algorithm suffer from artifacts due to the limited available angular tilt range. Algorithms such as the simultaneous iterative reconstruction technique (SIRT), which we have implemented in our laboratory, provide some improvements in the quality of 3D reconstructions but artifacts still persist. We are exploring other techniques by introducing some prior knowledge about the reconstructed volume by the application of regularization conditions.</p>
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<div class="field field--name-field-card-title field--type-string field--label-hidden field__item">Characterization of Bionanoparticles </div>
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<div class="field field--name-field-card-text field--type-text-long field--label-hidden field__item"><p>Bionanoparticles being developed for potential use in theranostic nanomedicine often have a hybrid structure that incorporates both organic and inorganic components. STEM and EFTEM provide unique information about the arrangement and proportions of the chemical constituents that are critical in controlling the function of such hybrid bionanoparticles. We are using these methods to characterize bionanoparticles containing a variety of moieties including MRI contrast agents, optical fluorescence probes, nanogold atomic clusters, carbon nanotubes carriers, as well as anti-cancer drugs such as doxorubicin.</p>
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<div class="field field--name-field-card-title field--type-string field--label-hidden field__item">SBF-SEM/FIB-SEM imaging and quantitative 3D analysis with machine learning</div>
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<div class="field__item"> <img loading="lazy" src="/sites/default/files/2025-01/semmachinelearning_0.jpg" width="428" height="373" alt="image generated by scanning electron microscope and machine learning" typeof="foaf:Image">
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<p>Using serial block-face (SBF) and focused ion beam (FIB) scanning electron microscopy (SEM) we acquire 3D cellular structures with axial resolutions of 30 nm and 5 nm, respectively. Unlike STEM and TEM tomography, using these complimentary imaging modalities we can obtain data from sample thicknesses up to ~1.2 mm (SBF-SEM) and ~50 mm (FIB-SEM), enabling comprehensive structural analysis of entire cells and substantial tissue regions. We employ machine learning algorithms for 3D modeling and segmentation. With these approaches, we are investigating the platelet distributions inside a mouse thrombi and how their activation states affect the clot formation.</p>
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