Electron microscope tomography of cells and tissues: studying the 3D structure of molecular machines at molecular resolution

Despite the large number of protein structures, we know little about their assembly into multi-protein complexes in cells. Such molecular machines are often transient, depend on their cellular environment, and therefore too complex, rare, and fragile to be purified, and therefore unsuitable for most structural techniques. I will illustrate the potential of EM tomography of cells and tissues with an emphasis on hearing machinery. I will describe the difficulties encountered, such as noise, macromolecular crowding and the complexity of the 3D data. I show how we currently overcome these challenges in order to achieve biological interpretations of the 3D cellular sceneries. We are using a variety of image processing analysis tools, mostly provided by others, such as bilateral filtering, gradient-based feature extraction, watershed immersion segmentation. We have started to employ skeletonization schemes to assess differences and similarities between these molecular machines in their respective cellular environment. In order to achieve the ultimate goal of understanding cells and tissues at a truly molecular level, we will need further improvements in automated image segmentation, template matching, as well as pattern recognition. Moreover, we hope to interest the audience in the development of an interactive, virtual reality-like 3D visualization and segmentation program, that may-make scientific analysis of cellular 3D volumes as easy and enjoyable as a video game. (Support: HFSPO (LT-0532); Agouron Institute/Jane Coffin Childs Fund; US-DOE (DE-A C03-76SF00098)).