Stagg Scott M, Lander Gabriel C, Pulokas James, Fellmann Denis, Cheng Anchi, Quispe Joel D, Mallick Satya P, Avila Radomir M, Carragher Bridget, Potter Clinton S
The National Resource for Automated Molecular Microscopy, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Struct Biol. 2006 Sep;155(3):470-81. doi: 10.1016/j.jsb.2006.04.005. Epub 2006 May 22.
One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images of macromolecular specimens embedded in vitreous ice. In this article, we demonstrate the potential of the Leginon system for high-throughput data acquisition by describing an experiment in which we acquired images of more than 280,000 particles of GroEL in a single 25 h session at the microscope. We also demonstrate the potential for an automated pipeline for molecular microscopy by showing that these particles can be subjected to completely automated procedures to reconstruct a three-dimensional (3D) density map to a resolution better than 8 A. In generating the 3D maps, we used a variety of metadata associated with the data acquisition and processing steps to sort and select the particles. These metadata provide a number of insights into factors that affect the quality of the acquired images and the resulting reconstructions. In particular, we show that the resolution of the reconstructed 3D density maps improves with decreasing ice thickness. These data provide a basis for assessing the capabilities of high-throughput macromolecular microscopy.
我们开发自动化电子显微镜数据采集系统Leginon的目标之一，是提高获取包埋在玻璃态冰中的大分子标本低剂量图像过程的易用性和通量。在本文中，我们通过描述一项实验来展示Leginon系统进行高通量数据采集的潜力，在该实验中，我们在显微镜下单次25小时的实验过程中采集了超过280,000个GroEL颗粒的图像。我们还通过表明这些颗粒可以经过完全自动化的程序来重建分辨率优于8埃的三维（3D）密度图，展示了分子显微镜自动化流程的潜力。在生成3D图时，我们使用了与数据采集和处理步骤相关的各种元数据来分类和选择颗粒。这些元数据为影响所采集图像质量和最终重建结果的因素提供了许多见解。特别是，我们表明重建的3D密度图的分辨率随着冰厚度的减小而提高。这些数据为评估高通量大分子显微镜的能力提供了基础。
