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控制的，它可以校正连续倾斜角之间的样品漂移和散焦。 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。 我们已经实现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。 已经获得的初步结果显示了使用双荧光纳米金标记的抗体对染色质绝缘体体复合物内所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。 我们还开发了其他重要生物元素（如钙）的定量能量过滤TEM成像方法，这需要比磷映射更精确的复数散射校正。 钙分布的EFTEM成像正被应用于研究快速冷冻和冷冻切片神经元中钙的调节。 这种方法是补充能量色散X射线光谱和电子能量损失光谱menasspectroscopy使用聚焦探头在扫描透射电子显微镜（STEM）。 EFTEM可以分析更大的样品区域，但灵敏度较低。

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, which 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 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. Preliminary results have been obtained showing 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 visalized 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. We have also developed methods for quantitative energy-filtered TEM imaging of other important biological elements like calcium, which requires even more precise correction for plural scattering than does phosphorus mapping. EFTEM imaging of calcium distributions is being applied to study the regulation of calcium in rapidly frozen and cryosectioned neurons. This approach is complementary to energy-dispersive x-ray spectroscopy and electron energy-loss spectroscopy menasurements using a focused-probe in the scanning transmission electron microscope (STEM). EFTEM enables much larger specimen areas to be analyzed but with lower sensitivity.