Energy-Filtered Electron Tomography

我们已经开发了一种称为定量电子光谱断层扫描（QuEST）的技术，用于成像细胞中特定化学元素的三维分布。利用300 kV场发射透射电子显微镜（TEM）和先进的成像滤光片，采集了一系列样品倾斜角度范围内的二维元素图。采集是通过灵活的计算机脚本控制，使校正标本漂移和离焦之间的连续倾斜角度。投影的二维元素分布是通过在能量损失谱中获取特征核心边缘上下的图像并减去每个像素的外推背景强度来获得的。我们实现并测试了一种双轴同步迭代重建技术（SIRT）来重建三维元素分布。通过应用考虑复数非弹性散射的厚度校正算法，并结合核壳电子激发的散射截面，我们已经表明，可以根据每体素的原子数来量化元素分布。利用相关光学显微镜和3-D磷成像技术，科学家们正在进行实验，以绘制DNA在细胞核特定区域的分布，其中大分子复合物参与基因调控。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内含有的特定蛋白质进行成像的可行性。利用荧光标记在光学显微镜下对蛋白质进行跟踪，然后利用扫描透射电子显微镜（STEM）模式下的电子断层扫描对金纳米颗粒标签进行三维可视化。然后利用电子瞬变电磁法断层扫描确定了绝缘体复合物附近DNA的分布。

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.