Large-Scale Electron Tomography of Cells Using SerialEM and IMOD

Abstract

We have developed methods to compute tomographic reconstructions of relatively large areas and volumes of sectioned cells using SerialEM and IMOD. The SerialEM program has emerged as a major tool for automated acquisition of tilt series for electron tomography [1]. This program contains a number of unique features, including a prediction algorithm that allows rapid image acquisition, and it can also routinely acquire tilt series from montaged images. The IMOD package contains alignment methods that correct for the non-uniform changes that occur over a large specimen area during data collection [1, 2]. In addition, the IMOD package offers tools for aligning and stacking tomograms from serial sections, allowing larger volumes to be reconstructed. Until recently practical considerations, such as constraints on electronic image shift, have limited the size of the area that could be reconstructed using ordinary montaging to ~10 × 10 µm. The desire to study areas larger than this has spurred the development of methods for stitching together laterally adjacent tomograms, referred to as super-montaging. In this chapter, we describe the steps involved in performing large-scale tomography including super-montaging, using the human mitotic spindle as an example.