Electron Tomography Of Cellular Structures

我们在配备柱后能量过滤器的数字透射电子显微镜上实现了电子断层扫描技术，以确定超分子组装和亚细胞细胞器的三维排列和结构。一系列能量过滤图像，每个包含1024 x 1204像素，在60到80个不同的倾斜角度，从-80度到+80度，使用冷却CCD相机获得。这些图像序列使用IMOD程序（科罗拉多大学）进行分析，以获得三维重建。金颗粒被吸附到切片上，以促进每个系列中的图像对齐。利用电子断层扫描技术，对高压冷冻、冷冻取代和重金属染色制备的大鼠脑神经元突触后密度（psd）中蛋白质的三维排列进行了研究。与psd相关的结构包括CAM激酶II，支架蛋白以及细胞骨架纤维。

We have implemented the technique of electron tomography in a digital transmission electron microscope equipped with a post-column energy-filter in order to determine the three-dimensional arrangement and structure of supramolecular assemblies and subcellular cellular organelles. Series of energy-filtered images each containing 1024 x 1204 pixels are acquired at between sixty and eighty different tilt angles from -80 degrees to +80 degrees using a cooled CCD camera. These image series are analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. Gold particles are adsorbed onto the sections to facilitate alignment of the images in each series. 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 extend the capabilities of electron tomography, we have developed a new technique enabling three-dimensional microanalysis based on core-excitation images. Using this approach, we have shown that it is possible to map the phosphorus in ribosomes in high-pressure frozen and freeze-substituted preparations of C. elegans. The method can distinguish between protein and nucleic acid in unstained specimen and might therefore find applications in the study of the cell nucleus.