Energy-Filtered Electron Microscopy and Electron Spectroscopic Imaging

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We have developed the technique of quantitative electron spectroscopic tomography (QuEST) for imaging the three-dimensional distribution of specific chemical elements in cells. A field-emission transmission electron microscope (TEM) operating at an accelerating voltage of 300 kV and equipped with an advanced imaging filter is used to collect a series of 2-D elemental maps for a range of specimen tilt angles. Acquisition is controlled by means of flexible computer scripts that enable correction for specimen drift and defocus between successive tilt angles. Projected 2-D elemental distributions are obtained by acquiring images above and below characteristic core-edges in the energy-loss spectrum and by subtracting the extrapolated background intensity at each pixel. We have implemented and tested a dual-axis simultaneous iterative reconstruction technique (SIRT) to reconstruct the 3-D elemental distribution. By applying a thickness correction algorithm that takes into account plural inelastic scattering, and by incorporating scattering cross sections for excitation of core-shell electrons, we have shown that it is possible to quantify the elemental distributions in terms of the number of atoms per voxel. By using correlative light microscopy and 3-D phosphorus imaging, experiments are in progress to map the distribution of DNA in specific domains of cell nuclei, where macromolecular complexes are involved in regulation of genes. We have demonstrated the feasibility of using a dual fluoro-nanogold labeled antibody to image specific proteins contained within the chromatin insulator body complex. The proteins can be tracked in the optical microscope using the fluorescence tag, after which the gold nanoparticle tags can be visualized in 3D using electron tomography in the scanning transmission electron microscope (STEM) mode. Then EFTEM tomography is used to determine the distribution of DNA in the vicinity of the insulator body complex. The application of the QuEST technique is limited by radiation damage, which has the potential to alter the elemental composition as well as the specimen morphology, and we have performed a systematic study to determine the effect of electron dose. Electron tomograms obtained from unstained high-pressure frozen and freeze-substituted sections of Caenorhabditis elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins. A new-generation Gatan Dual EELS imaging filter on our FEI Tecnai TF30 transmission electron microscope is now providing much higher sensitivity for elemental analysis than has been previously possible. We have used this system to map and quantify the distributions of ferritin in differentiating erythroblasts. Data acquired with the dual-EELS mode enabled precise calibration of the energy losses throughout hyperspectral images, as well as determination of the number of iron atoms per pixel in elemental maps. We have examined ex vivo erythroid cultures of primary CD34+ cells, for accumulation of iron during different stages of development. The iron maps showed that punctate particles in vesicles surrounding mitochondria contained between 2,000 and 4,000 Fe atoms, consistent with cores of ferritin molecules. The number of ferritin cores was highest for cells incubated ex vivo for 14 days, after which the number of ferritin molecules was reduced again due to the formation of heme. Our results indicate that, in cultured differentiating erythroblasts, iron is accumulated and stored as Fe(III) in the earlier stages of erythropoiesis. We have also applied STEM-EELS spectrum imaging to investigate the composition of gravity sensors in a model organism of interest to neuroscientists, Trichoplax adhaerens, which is a simple animal of the ancient phylum Placozoa. Trichoplax has only six cell types, one of which is the crystal cell, the least numerous cell type. Crystal cells are arrayed around the perimeter of the animal and each contains a birefringent crystal. Crystal cells resemble lithocytes in other animals and electron microscopy revealed crystal cell contacts with fiber cells and epithelial cells but these contacts lacked features of synapses. STEM-EEELS spectrum imaging at the carbon K edge and calcium L2,3 edge showed that crystals consist of the aragonite form of calcium carbonate. Calcium is present in the statoliths of many cnidarians, but it rarely binds carbonate anions, with medusae deploying calcium phosphate or calcium sulfate as a major component of their statoliths. Our results show neither phosphorus nor sulfur in the lithocytes of Trichoplax, whereas the statoliths of the simple flatworm Acoela, whose lithocytes are similar to crystal cells of Trichoplax, are composed of calcium phosphate (apatite). Interestingly, statoliths of the vast majority of higher animals consist of calcium carbonate in mammals as well as mollusks and teleost fish. It therefore seems likely, given the morphological and compositional diversity of statocysts in different animal taxa, that gravity receptors independently evolved more than once, even in different species of flatworms.