Energy-Filtered Electron Microscopy and Electron Spectroscopic Imaging

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

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 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 obtained from human CD34(+) cells from peripheral blood via leukapheresis. A crucial step in erythropoiesis, the labile iron pool and its transport to mitochondria for heme production, is not well understood. We have applied a dual 3D imaging and spectroscopic technique, based on scanned electron probes: (1) scanning transmission electron microscopy/electron energy loss spectroscopy (STEM/EELS), and (2) serial block-face electron microscopy (SBEM), to measure distributions of ferritin iron-storage protein in ex vivo human erythropoietic stem cells, and to determine how those distributions change during terminal differentiation. After seven days of differentiation, the cells display a highly specialized architecture of organelles with anchored clustering of mitochondria and massive accumulation of Fe3+ in loaded ferritin cores localized to lysosomal storage depots, providing an iron source for heme production. Macrophages are not present in our ex vivo cultures, so they cannot be the source of the ferritin. Our measurements indicate that lysosomal iron depots are required by developing reticulocytes while terminally differentiating and continuing to produce heme and globin, which assemble and concentrate to fill the cytoplasm after much of the cellular machinery is expelled (M. Aronova et al., iScience, 2021). Data acquired with the dual-EELS mode have 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. The iron maps reveal 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.