Automated Data Collection with a Tecnai 12 Electron Microscope: Applications for Molecular Imaging by Cryomicroscopy

Abstract

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.