Energy-Filtered Electron Microscopy and Electron Spectroscopic Imaging

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

A Gatan Quantum Dual-EELS imaging filter interfaced to a Thermo/FEI Tecnai TF30 transmission electron microscope provides a high sensitivity for elemental analysis of biological structures through STEM-EELS hyperspectral imaging, as well as by energy-filtered electron spectroscopic imaging. 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.