Energy-Filtered Electron Tomography

我们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。一个300千伏的场发射透射电子显微镜（TEM）配备了先进的成像过滤器是用来收集一系列的2-D元素的标本倾斜角的范围内的地图。采集是通过灵活的计算机脚本，使连续倾斜角之间的样品漂移和散焦校正控制。 投影的2-D元素分布是通过在能量损失谱中获取特征核心边缘上方和下方的图像并通过在每个像素处减去外推的背景强度来获得的。 我们已经实施并测试了双轴同步迭代重建技术（SIRT）重建的3-D元素分布。通过应用厚度校正算法，考虑到多个非弹性散射，并通过将散射截面的核壳电子的激发，我们已经表明，它是可能的，以量化的元素分布在每体素的原子数。 通过使用相关光学显微镜和3-D磷成像，正在进行实验以绘制DNA在细胞核特定区域中的分布，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荧光纳米金标记的抗体来成像染色质绝缘体复合物内包含的特定蛋白质的可行性。 可以使用荧光标签在光学显微镜中跟踪蛋白质，之后可以在扫描透射电子显微镜（STEM）模式中使用电子断层扫描在3D中可视化金纳米颗粒标签。 然后EFTEM断层扫描被用来确定在附近的绝缘体复合体的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试样形态，我们已经进行了系统的研究，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中获得的电子断层图像表明，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.