Energy-Filtered Electron Microscopy and Electron Spectroscopic Imaging

能量过滤电子显微镜和电子光谱成像

• 批准号: 10701550
• 负责人: Richard Leapman
• 金额: $ 76.26万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10701550
• 关键词: 3-Dimensional; Architecture; Biological; Blood Cells; CD34 gene; Calibration; Cells; Chemicals; Cytoplasm; Data; Electron Microscope; Electron Microscopy; Electron energy loss spectroscopy; Electrons; Elements; Energy-Filtering Transmission Electron Microscopy; Erythroblasts; Erythroid Progenitor Cells; Erythropoiesis; Face; Ferritin; Globin; Heme; Human; Image; Incubated; Iron; Leukapheresis; Maps; Measurement; Measures; Mitochondria; Organelles; Production; Proteins; Reticulocytes; Scanning; Scanning Electron Microscopy; Source; Structure; System; Techniques; Three-Dimensional Imaging; Transmission Electron Microscopy; Vesicle; base; electron energy; macrophage; particle; quantum; spectroscopic imaging; transmission process

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.

Gatan Quantum Dual-EELS 成像滤光片与 Thermo/FEI Tecnai TF30 透射电子显微镜连接，通过 STEM-EELS 高光谱成像以及能量过滤电子光谱成像，为生物结构的元素分析提供高灵敏度。我们使用该系统来绘制和量化铁蛋白在分化成红细胞中的分布，该成红细胞是通过白细胞分离术从外周血中获得的人 CD34(+) 细胞获得的。红细胞生成过程中的一个关键步骤，即不稳定的铁池及其转运至线粒体以产生血红素的过程，目前尚不清楚。我们应用了基于扫描电子探针的双重 3D 成像和光谱技术：(1) 扫描透射电子显微镜/电子能量损失光谱 (STEM/EELS) 和 (2) 串行块面电子显微镜 (SBEM)，以测量离体人类红细胞生成干细胞中铁蛋白储铁蛋白的分布，并确定这些分布在终末分化过程中如何变化。经过 7 天的分化，细胞显示出高度专业化的细胞器结构，其中有锚定的线粒体簇，并且在位于溶酶体储存库的负载铁蛋白核心中大量积累 Fe3+，为血红素生产提供铁源。巨噬细胞不存在于我们的离体培养物中，因此它们不能是铁蛋白的来源。我们的测量表明，网织红细胞发育过程中需要溶酶体铁库，同时最终分化并继续产生血红素和球蛋白，在大部分细胞机器被排出后，它们组装并集中以填充细胞质（M. Aronova 等人，iScience，2021） ）。通过双 EELS 模式获取的数据可以精确校准整个高光谱图像的能量损失，并确定元素图中每个像素的铁原子数量。铁图显示线粒体周围囊泡中的点状颗粒含有 2,000 至 4,000 个 Fe 原子，与铁蛋白分子的核心一致。离体培养14天的细胞中铁蛋白核心的数量最高，之后由于血红素的形成，铁蛋白分子的数量再次减少。我们的结果表明，在培养的分化成红细胞中，铁在红细胞生成的早期阶段以 Fe(III) 的形式积累和储存。

项目成果

Elemental mapping by electron energy loss spectroscopy in biology.

生物学中电子能量损失光谱的元素图谱。

• 发表时间: 2013
• 作者: Aronova, Maria A;Leapman, Richard D
• 通讯作者: Leapman, Richard D

Development of Electron Energy Loss Spectroscopy in the Biological Sciences.

电子能量损失光谱在生物科学中的发展。

• 发表时间: 2012-01
• 影响因子: 5
• 作者: Aronova, M A;Leapman, R D
• 通讯作者: Leapman, R D

Manipulating the power of an additional phase: a flower-like Au-Fe3O4 optical nanosensor for imaging protease expressions in vivo.

操纵附加相的功率：花状 Au-Fe3O4 光学纳米传感器，用于体内蛋白酶表达成像。

• 发表时间: 2011-04-26
• 影响因子: 17.1
• 作者: Xie, Jin;Zhang, Fan;Aronova, Maria;Zhu, Lei;Lin, Xin;Quan, Qimeng;Liu, Gang;Zhang, Guofeng;Choi, Ki;Kim, Kwangmeyung;Sun, Xiaolian;Lee, Seulki;Sun, Shouheng;Leapman, Richard;Chen, Xiaoyuan
• 通讯作者: Chen, Xiaoyuan

Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses.

含有文石晶体的细胞介导粘性毛盘菌（Placozoa）对重力的反应，这是一种缺乏神经元和突触的动物。

• 发表时间: 2018
• 影响因子: 3.7
• 作者: Mayorova, Tatiana D;Smith, Carolyn L;Hammar, Katherine;Winters, Christine A;Pivovarova, Natalia B;Aronova, Maria A;Leapman, Richard D;Reese, Thomas S
• 通讯作者: Reese, Thomas S

Layer-specific variation of iron content in cerebral cortex as a source of MRI contrast.

大脑皮层铁含量的层特异性变化作为 MRI 对比的来源。

• 发表时间: 2010-02-23
• 影响因子: 11.1
• 作者: Fukunaga, Masaki;Li, Tie;van Gelderen, Peter;de Zwart, Jacco A;Shmueli, Karin;Yao, Bing;Lee, Jongho;Maric, Dragan;Aronova, Maria A;Zhang, Guofeng;Leapman, Richard D;Schenck, John F;Merkle, Hellmut;Duyn, Jeff H
• 通讯作者: Duyn, Jeff H