Electron Tomography Of Cellular Structures

我们已经实施了自动电子断层扫描技术，在300千伏场发射TEM配备了柱后能量过滤器，以确定超分子组件和亚细胞细胞器的三维排列。使用冷却的CCD照相机在-70度至+70度的样品倾斜范围内和对于旋转90度的双倾斜轴，获得一系列未过滤或能量过滤的数字图像，每个图像包含2048 × 2048像素。使用IMOD程序（科罗拉多大学）分析这些图像系列以获得三维重建。 我们已经应用电子断层扫描，以确定三维排列的蛋白质在突触后密度（PSD）的培养神经元从大鼠脑制备的高压冷冻，冷冻置换和重金属染色。与PSD相关的感兴趣的结构包括CAM激酶II、支架蛋白以及细胞骨架纤维。 为了补充NIDDK研究人员正在进行的研究，以了解尼曼-皮克C型疾病中发生的内吞转运途径的缺陷，我们正在确定培养的成纤维细胞和CHO细胞中含有野生型或突变型NPC蛋白的内体囊泡和内体小管的细胞超微结构。重建显示多层小管，似乎与先前在标记细胞的荧光显微镜下观察到的晚期内体小管相关。

We have implemented the technique of automated electron tomography in a 300 kV field-emission TEM equipped with a post-column energy-filter to determine the three-dimensional arrangement of supramolecular assemblies and subcellular cellular organelles. Series of unfiltered or energy-filtered digital images each containing 2048 x 2048 pixels were acquired using a cooled CCD camera over a specimen tilt range from -70 degrees to +70 degrees and for dual tilt axes rotated by 90 degrees. These image series were analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. We have applied electron tomography to determine the three-dimensional arrangement of proteins in post-synaptic densities (PSDs) of cultured neurons from rat brain prepared by high-pressure freezing, freeze-substitution and heavy metal staining. The structures of interest associated with the PSDs include CAM kinase II, scaffolding proteins as well as cytoskeletal fibers. To complement studies being conducted by NIDDK investigators to understand defects in the endocytic trafficking pathway that occur in Niemann-Pick Type C Disease, we are determining the cellular ultrastructure of endosomal vesicles and endosomal tubules in cultured fibroblasts and CHO cellls containing wild-type or mutant NPC protein. The reconstructions show multi-lamellar tubules that appear to correlate with late endosomal tubules previously observed in fluorescent light microscopy of labeled cells.