Energy-Filtered Electron Tomography

能量过滤电子断层扫描

• 批准号: 8340619
• 负责人: Richard Leapman
• 金额: $ 11.48万
• 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/8340619
• 关键词: 3-Dimensional; Accounting; Algorithms; Anatomy; Antibodies; Architecture; Autopsy; Brain; Caenorhabditis elegans; Cell Nucleus; Cells; Characteristics; Chemicals; Chromatin; Complex; Computers; DNA; Data; Dose; Electron Microscope; Electron Microscopy; Electron energy loss spectroscopy; Electrons; Elements; Energy-Filtering Transmission Electron Microscopy; Ferritin; Fluorescence; Freezing; Gene Expression; Gene Expression Regulation; Gold; Higher Order Chromatin Structure; Human; Image; Imagery; Indium; Iron; Label; Macromolecular Complexes; Magnetic Resonance Imaging; Magnetism; Maps; Measures; Microscope; Morphology; Nitrogen; Nucleic Acids; Occipital lobe; Optics; Pattern; Phosphorus; Property; Proteins; Radiation; Scanning; Series; Source; Specimen; Techniques; Testing; Thick; Tissues; Tomogram; Variant; base; brain tissue; electron tomography; flexibility; gray matter; imaging modality; improved; in vivo; interest; light microscopy; nanoGold; nanoparticle; optical imaging; particle; pressure; reconstruction; research study; tomography; transmission process; two-dimensional; voltage

We have developed a technique called quantitative electron spectroscopic tomography (QuEST) for imaging the three-dimensional distribution of specific chemical elements in cells. A 300 kV field-emission transmission electron microscope (TEM) 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. We have also applied energy-filtered electron microscopy to elucidate contrast mechanisms in brain MRI. Recent advances in high field MRI have dramatically improved the visualization of human brain anatomy in vivo. For example, in cortical gray matter, strong contrast variations have been observed that appear to reflect the local laminar architecture. This contrast has been attributed to subtle variations in the magnetic properties of brain tissue, possibly reflecting varying iron content. To establish the origin of this contrast, MRI data of postmortem brain in the region of the occipital cortex were correlated with optical images, electron micrographs and electron spectroscopic images. The results showed that iron is distributed over laminae in a pattern that correlates with the dominant source of the observed MRI contrast in the line of Gennari. In particular, the iron was contained in dense particles containing approximately 1,700 Fe atoms, which could be identified as ferritin cores.

我们开发了一种称为定量电子能谱断层扫描 (QuEST) 的技术，用于对细胞中特定化学元素的三维分布进行成像。配备先进成像滤光片的 300 kV 场发射透射电子显微镜 (TEM) 用于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节。 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。 我们还应用能量过滤电子显微镜来阐明脑 MRI 的对比机制。高场 MRI 的最新进展极大地改善了人脑体内解剖结构的可视化。例如，在皮质灰质中，观察到强烈的对比度变化，这似乎反映了局部层状结构。这种对比归因于脑组织磁性的细微变化，可能反映了铁含量的变化。为了确定这种对比的起源，将死后大脑枕叶皮质区域的 MRI 数据与光学图像、电子显微照片和电子能谱图像相关联。 结果表明，铁在椎板上的分布模式与在 Gennari 线中观察到的 MRI 对比度的主要来源相关。 特别是，铁包含在含有大约 1,700 个 Fe 原子的致密颗粒中，可以将其识别为铁蛋白核心。