We have developed methods for generating tomographic reconstructions of large cellular volumes efficiently and at relatively high resolution. One key component of these methods is the ability to capture images from our high voltage electron microscope (HVEM) with a sensitive lenscoupled CCD camera. to overcome the limited resolution and field of view of this 1k × 1K camera, we have used the image shift capability of the HVEM to develop automatic montaging of images from an array of positions, with about 10% overlap between adjacent positions. We have also developed automatic repositioning and autofocusing after each tilt of the specimen, so that it is now possible to acquire single-frame or montaged images at a series of tilt angles, with no operator intervention. with these capabilities, up to 2 GB of image data can be acquired in a single session.
Tilt series are processed with the IMOD software package developed in our laboratory, which now runs on a PC under Linux, as well as on SGI computers. Several features are notable for working with large data sets.