The Cellular and Supramolecular Structure and Function (CSSF) Section develops new methods based on electron microscopy and related techniques. Our aim is to expand knowledge about complex biological and disease processes, as well as to characterize morphologically the action of diagnostic markers and therapeutic agents in cells. The nanometer scale of biological electron microscopy lies between the realms of live-cell optical microscopy and atomic-scale structural tools that require extraction and purification of cellular components. Current research includes development of techniques for (1) determining the tertiary and quaternary structures of macromolecular assemblies, (2) visualizing 3D ultrastructure, (3) mapping the elemental composition of subcellular compartments quantitatively, and (4) studying bionanoparticles and their interactions with cells. We are applying these methods to structural biology, cellular biology, neurobiology, cancer biology, and nanomedicine.
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.
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.
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.
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.
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.
The Cellular and Supramolecular Structure and Function (CSSF) Section of LCIMB is located in Building 13, Room 3E63.
The laboratory has facilities for preparing tissues, cells, and isolated macromolecular assemblies for electron microscopy, either in the CSSF Section or in the neighboring NIH-wide Electron Microscopy Shared Resource. Preparative equipment includes a Baltec HPM10 high-pressure freezing machine, FEI vitrobot for freezing EM grids, Leica UCT/FCS cryo-ultramicrotome, Leica EM UC6 ultramicrotome, Leica EM/AFS2 freeze-substitution system, EMS and Edwards 306 carbon evaporators.
The laboratory’s dedicated electron microscope is an FEI Tecnai TF30 TEM operated at an accelerating voltage of 300 kV and equipped with a field-emission source. The instrument is also equipped with a Gatan Tridiem imaging filter, two 2k x 2k pixel Gatan Ultrascan cooled CCD cameras, a Fischione HAADF detector, Fischione dual-axis tomography holder, Gatan cryo-transfer tomography holder. The instrument is also has FEI and Gatan software packages for performing electron tomography, hyperspectral imaging, electron energy loss spectroscopy, and scanning transmission electron microscopy. Through the neighboring Electron Microscopy Shared Resource, the laboratory also has access to an FEI T12 TEM operating at an accelerating voltage of 120 kV, and equipped for electron tomography, and energy-dispersive x-ray spectroscopy. Also available are a Hitachi H4800 field-emission scanning electron microscope, and a Zeiss Sigma SEM equipped with a Gatan 3View serial blockface imaging system.