Electron Tomography Of Cellular Structures

细胞结构的电子断层扫描

• 批准号: 6837245
• 负责人: Richard D Leapman
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6837245
• 关键词: animal tissue; bioimaging /biomedical imaging; brain imaging /visualization /scanning; calmodulin dependent protein kinase; cell component structure /function; charge coupled device camera; cytoskeletal proteins; gold; intracellular; laboratory rat; molecular assembly /self assembly; neurons; phosphotransferases; protein structure; ribosomes; technology /technique development; three dimensional imaging /topography; tissue /cell culture; tomography; transmission electron microscopy

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.

我们在配备柱后能量滤波器的数字透射电子显微镜中实施了电子断层扫描技术，以确定超分子组装体和亚细胞细胞器的三维排列和结构。使用冷却 CCD 相机，以 -80 度到 +80 度之间的 60 到 80 个不同倾斜角度采集一系列能量过滤图像，每个图像包含 1024 x 1204 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。金颗粒被吸附到切片上，以促进每个系列中图像的对齐。我们应用电子断层扫描来确定通过高压冷冻、冷冻替代和重金属染色制备的大鼠脑培养神经元的突触后密度（PSD）中蛋白质的三维排列。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。 为了扩展电子断层扫描的功能，我们开发了一种新技术，能够基于核心激发图像进行三维微分析。使用这种方法，我们已经证明可以绘制线虫高压冷冻和冷冻替代制剂中核糖体中的磷图谱。该方法可以区分未染色样本中的蛋白质和核酸，因此可能在细胞核的研究中得到应用。