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: 10011332
• 负责人: Richard Leapman
• 金额: $ 3.76万
• 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/10011332
• 关键词: 3-Dimensional; Algorithms; Animal Model; Animals; Anions; Antibodies; Apatites; Aragonite; Binding; CD34 gene; Caenorhabditis elegans; Calcium; Calcium Carbonate; Calcium Sulfate; Calibration; Carbon; Carbonates; Cell Nucleus; Cells; Characteristics; Chemicals; Chromatin; Cnidaria; Complex; Computers; Crystallization; DNA; Data; Development; Dimensions; Dose; Electron Microscope; Electron Microscopy; Electrons; Elements; Energy-Filtering Transmission Electron Microscopy; Epithelial Cells; Erythroblasts; Erythroid; Erythroid Progenitor Cells; Erythropoiesis; Face; Ferritin; Fluorescence; Force of Gravity; Freeze Substitution; Gene Expression Regulation; Generations; Goals; Heme; Higher Order Chromatin Structure; Image; Incubated; Iron; Label; Macromolecular Complexes; Mammals; Maps; Measures; Microscope; Mitochondria; Morphology; Nitrogen; Nucleic Acids; Optics; Phosphorus; Platyhelminths; Proteins; Radiation induced damage; Reticulocytes; Scanning; Scanning Electron Microscopy; Series; Specimen; Sulfur; Synapses; System; Techniques; Testing; Thick; Tomogram; Vesicle; calcium phosphate; cell type; electron energy; electron tomography; experimental study; fiber cell; flexibility; improved; interest; light microscopy; nanoGold; particle; pressure; receptor; reconstruction; sensor; spectrograph; spectroscopic imaging; teleost fish; tomography; transmission process; voltage

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.

我们开发了定量电子能谱断层扫描（QuEST）技术，用于对细胞中特定化学元素的三维分布进行成像。使用在 300 kV 加速电压下运行并配备先进成像滤波器的场发射透射电子显微镜 (TEM) 来收集一系列样品倾斜角度的二维元素图。采集通过灵活的计算机脚本进行控制，可以校正连续倾斜角度之间的样本漂移和散焦。 通过采集能量损失谱中特征核心边缘上方和下方的图像并减去每个像素处的外推背景强度，获得投影的二维元素分布。 我们实施并测试了双轴同时迭代重建技术 (SIRT) 来重建 3D 元素分布。通过应用考虑多重非弹性散射的厚度校正算法，并通过合并用于激发核壳电子的散射截面，我们已经证明可以根据每个体素的原子数量来量化元素分布。 通过使用相关光学显微镜和 3-D 磷成像，实验正在进行中，以绘制细胞核特定区域中 DNA 的分布图，其中大分子复合物参与基因的调控。 我们已经证明了使用双氟纳米金标记抗体对染色质绝缘体复合物中包含的特定蛋白质进行成像的可行性。 可以使用荧光标签在光学显微镜中追踪蛋白质，之后可以使用扫描透射电子显微镜 (STEM) 模式下的电子断层扫描以 3D 方式可视化金纳米颗粒标签。 然后使用 EFTEM 断层扫描确定绝缘体复合体附近 DNA 的分布。 QuEST 技术的应用受到辐射损伤的限制，辐射损伤有可能改变元素组成以及样品形态，我们进行了系统研究以确定电子剂量的影响。 从秀丽隐杆线虫未染色的高压冷冻和冷冻替代切片获得的电子断层图表明，获得有用的 3D 磷和氮图是可行的，从而揭示有关核酸和蛋白质的亚细胞分布的定量信息。 我们的 FEI Tecnai TF30 透射电子显微镜上的新一代 Gatan Dual EELS 成像滤光片现在可为元素分析提供比以前更高的灵敏度。 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。使用双 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。我们检查了原代 CD34+ 细胞的离体红系培养物，以了解不同发育阶段铁的积累。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。 我们还应用 STEM-EELS 光谱成像来研究神经科学家感兴趣的模型生物体中重力传感器的组成，Trichoplax adhaerens 是古代扁动物门的一种简单动物。 Trichoplax只有六种细胞类型，其中一种是晶体细胞，是数量最少的细胞类型。晶体细胞排列在动物的周围，每个细胞都包含一个双折射晶体。晶体细胞类似于其他动物中的石细胞，电子显微镜显示晶体细胞与纤维细胞和上皮细胞接触，但这些接触缺乏突触特征。碳 K 边缘和钙 L2,3 边缘的 STEM-EELS 光谱成像表明晶体由文石形式的碳酸钙组成。钙存在于许多刺胞动物的平衡石中，但很少与碳酸根阴离子结合，水母将磷酸钙或硫酸钙作为其平衡石的主要成分。我们的结果显示，毛盘虫的石细胞中既不含磷，也不含硫，而简单扁虫Acoela的石细胞与毛盘虫的晶体细胞相似，其石质细胞由磷酸钙（磷灰石）组成。有趣的是，绝大多数高等动物的平衡石由哺乳动物以及软体动物和硬骨鱼的碳酸钙组成。因此，考虑到不同动物类群中静囊的形态和组成多样性，重力受体似乎可能不止一次独立进化，甚至在不同种类的扁虫中也是如此。