Automated data collection with a tecnai 12 electron microscope: Applications for molecular imaging by cryomicroscopy
Automated data collection with a tecnai 12 electron microscope: Applications for molecular imaging by cryomicroscopy
复制标题
DOI：
10.1006/jsbi.2001.4404
复制
发表时间：
2001-09-01
影响因子：
3
通讯作者：
Subramaniam, S
中科院分区：
文献类型：
--
作者：
Zhang, PJ;Beatty, A;Subramaniam, S
在高分辨率生物电子显微镜中，收集大量高质量显微照片的速度是整个结构测定过程中的限速步骤。加速数据收集的方法可能非常有用，特别是在大型多蛋白质和蛋白质-核酸复合物的“单分子”显微镜中，其中需要平均数千个单独的分子图像以确定三维结构。为此，我们报告的开发自动化低剂量图像采集程序Tecnai 12电子显微镜上使用的脚本功能，可在显微镜计算机。在自动化的最低水平，用户需要选择要成像的感兴趣区域。然后自动执行所有后续的图像采集步骤，以在满足用户指定的样品台漂移率限制的条件下，以所需的散焦值在胶片或CCD上记录高分辨率图像。在最高的自动化水平下，自动执行最佳网格正方形和适合成像的最佳区域的确定。中等水平的自动化也是可用的，其中用户可以手动指定最有希望的网格正方形，并将在这些网格正方形中找到最佳孔的过程留给显微镜计算机。我们还表明，所有的步骤，随后插入的标本在显微镜中可以进行远程连接到显微镜的计算机通过互联网。这两个功能都是使用Windows NT和基于Web的工具实现的，并为任何位置的任何Tecnai显微镜提供自动数据收集工具。
In high-resolution biological electron microscopy, the speed of collection of large numbers of high-quality micrographs is a rate-limiting step in the overall process of structure determination. Approaches to speed up data collection can be very useful, especially in "single-molecule" microscopy of large multiprotein and protein-nucleic acid complexes, where many thousands of individual molecular images need to be averaged to determine the three-dimensional structure. Toward this end, we report the development of automated low-dose image acquisition procedures on a Tecnai 12 electron microscope using the scripting functionality available on the microscope computer. At the lowest level of automation, the user is required to select regions of interest that are to be imaged. All subsequent steps of image acquisition are then carried out automatically to record high-resolution images on either film or CCD, at desired defocus values, under conditions that satisfy user-specified limits for drift rates of the specimen stage. At the highest level of automation, determination of the best grid squares and the best regions suitable for imaging are carried out automatically. A medium level of automation is also available in which the user can designate the most promising grid squares manually and leave the process of finding the best holes in those grid squares to the microscope computer. We also show that all steps subsequent to insertion of the specimen in the microscope can be carried out remotely by connecting to the microscope computer via the Internet. Both features are implemented using Windows NT and Web-based tools and provide tools for automated data collection on any Tecnai microscope from any location.