Energy-Filtered Electron Microscopy and Electron Spectroscopic Imaging

我们已经开发了定量电子光谱断层扫描（QuEST）成像的细胞中的特定化学元素的三维分布的技术。一个场发射透射电子显微镜（TEM）在300千伏的加速电压下工作，并配备了先进的成像过滤器是用来收集一系列的2-D元素图的范围内的标本倾斜角。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。 我们的FEI Tecnai TF 30透射电子显微镜上的新一代Gatan Dual EELS成像滤光片现在为元素分析提供了比以前更高的灵敏度。 我们已经使用这个系统来映射和量化的铁蛋白在分化成红细胞中的分布。使用双EELS模式获得的数据可以精确校准整个高光谱图像的能量损失，以及确定元素图中每个像素的铁原子数量。我们已经检查了原代CD 34+细胞的离体红系培养物，在不同的发育阶段铁的积累。铁图谱显示，线粒体周围囊泡中的点状颗粒含有2,000至4,000个铁原子，与铁蛋白分子的核心一致。离体孵育14天的细胞的铁蛋白核心的数量是最高的，之后由于血红素的形成，铁蛋白分子的数量再次减少。我们的研究结果表明，在培养的分化成红细胞，铁积累和存储为Fe（III）在红细胞生成的早期阶段。 我们还应用STEM-EELS光谱成像来研究神经科学家感兴趣的模式生物Trichoplax adhaerens中重力传感器的组成，Trichoplax adhaerens是一种简单的古老动物门的动物。Trichoplax只有六种细胞类型，其中之一是晶体细胞，数量最少的细胞类型。晶体细胞排列在动物的周围，每个细胞都包含一个双折射晶体。晶体细胞类似于其他动物的石细胞，电子显微镜显示晶体细胞与纤维细胞和上皮细胞接触，但这些接触缺乏突触的特征。在碳K边缘和钙L2，3边缘处的STEM-EEELS光谱成像显示晶体由碳酸钙的文石形式组成。钙存在于许多刺胞动物的耳石中，但它很少结合碳酸根阴离子，水母部署磷酸钙或硫酸钙作为其耳石的主要成分。我们的研究结果表明，Trichoplax的石细胞中既不含磷也不含硫，而简单的扁虫Acoela的平衡石，其石细胞类似于Trichoplax的晶体细胞，由磷酸钙（磷灰石）组成。有趣的是，绝大多数高等动物的耳石由碳酸钙组成，哺乳动物以及软体动物和硬骨鱼。因此，考虑到不同动物类群平衡囊的形态和组成多样性，重力感受器独立进化不止一次，即使在不同种类的扁形虫中也是如此。

We have developed the technique of quantitative electron spectroscopic tomography (QuEST) for imaging the three-dimensional distribution of specific chemical elements in cells. A field-emission transmission electron microscope (TEM) operating at an accelerating voltage of 300 kV and 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. A new-generation Gatan Dual EELS imaging filter on our FEI Tecnai TF30 transmission electron microscope is now providing much higher sensitivity for elemental analysis than has been previously possible. We have used this system to map and quantify the distributions of ferritin in differentiating erythroblasts. Data acquired with the dual-EELS mode enabled precise calibration of the energy losses throughout hyperspectral images, as well as determination of the number of iron atoms per pixel in elemental maps. We have examined ex vivo erythroid cultures of primary CD34+ cells, for accumulation of iron during different stages of development. The iron maps showed that punctate particles in vesicles surrounding mitochondria contained between 2,000 and 4,000 Fe atoms, consistent with cores of ferritin molecules. The number of ferritin cores was highest for cells incubated ex vivo for 14 days, after which the number of ferritin molecules was reduced again due to the formation of heme. Our results indicate that, in cultured differentiating erythroblasts, iron is accumulated and stored as Fe(III) in the earlier stages of erythropoiesis. We have also applied STEM-EELS spectrum imaging to investigate the composition of gravity sensors in a model organism of interest to neuroscientists, Trichoplax adhaerens, which is a simple animal of the ancient phylum Placozoa. Trichoplax has only six cell types, one of which is the crystal cell, the least numerous cell type. Crystal cells are arrayed around the perimeter of the animal and each contains a birefringent crystal. Crystal cells resemble lithocytes in other animals and electron microscopy revealed crystal cell contacts with fiber cells and epithelial cells but these contacts lacked features of synapses. STEM-EEELS spectrum imaging at the carbon K edge and calcium L2,3 edge showed that crystals consist of the aragonite form of calcium carbonate. Calcium is present in the statoliths of many cnidarians, but it rarely binds carbonate anions, with medusae deploying calcium phosphate or calcium sulfate as a major component of their statoliths. Our results show neither phosphorus nor sulfur in the lithocytes of Trichoplax, whereas the statoliths of the simple flatworm Acoela, whose lithocytes are similar to crystal cells of Trichoplax, are composed of calcium phosphate (apatite). Interestingly, statoliths of the vast majority of higher animals consist of calcium carbonate in mammals as well as mollusks and teleost fish. It therefore seems likely, given the morphological and compositional diversity of statocysts in different animal taxa, that gravity receptors independently evolved more than once, even in different species of flatworms.