Energy-Filtered Electron Tomography

我们开发了一种称为定量电子光谱断层成像(QUE)的技术，用于成像细胞中特定化学元素的三维分布。利用一台配有先进成像滤光片的300KV场发射电子显微镜采集了一系列样品倾角范围内的二维元素图。采集通过灵活的计算机脚本进行控制，这些脚本可以对连续倾斜角度之间的样品漂移和散焦进行校正。通过获取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的外推背景强度来获得投影的2-D元素分布。我们已经实现并测试了一种双轴同时迭代重建技术(SIRT)来重建三维元素分布。通过应用考虑多次非弹性散射的厚度校正算法，以及结合核壳电子激发的散射截面，我们已经证明了用每个体素的原子数量来量化元素分布是可能的。利用相关光学显微镜和三维磷成像技术，正在进行绘制DNA在细胞核特定区域的分布的实验，其中大分子复合体参与了基因的调控。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体中包含的特定蛋白质进行成像的可行性。使用荧光标签可以在光学显微镜中跟踪蛋白质，然后可以在扫描透射电子显微镜(STEM)模式下使用电子断层扫描以3D方式可视化金纳米标签。然后使用EFTEM层析成像来确定DNA在绝缘体复合体附近的分布。 QUEST技术的应用受到辐射损伤的限制，这可能会改变样品的元素组成和形貌，我们已经进行了系统的研究，以确定电子剂量的影响。对秀丽线虫未染色的高压冷冻切片和冷冻置换切片的电子断层图像表明，获得有用的三维磷和氮图是可行的，从而揭示核酸和蛋白质的亚细胞分布的定量信息。

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.