2D and 3D Imaging of Entire Cells and Tissues at Macromolecular Resolution by Advanced Electron Microscopic Approaches
Manfred Auer (April 21, 2014)
Please install the Flash Plugin
All processes defining and sustaining life take place within cells or between cells, often mediated by multiprotein supramolecular complexes also known as macromolecular machines . While Structural Genomics and recently Cryo-EM have yielded ever-increasing insight into the overall shape of such macromolecular machines and the detailed mechanism of e.g. catalysis, we lack insight how these machines are organized and function within cells. We must determine their 3D organization, subcellular location (e.g. with respect to ultrastructural landmarks) and their interaction with other proteins, the cytoskeleton and organelles and any changes of these characteristics during embryonic development, as part of their physiological function or during pathogenesis.
Using various biological examples, including inner ear hair cells and related tissues central to hearing, mammary gland development and breast cancer, as well microbial communities, I will illustrate the power of modern 2D and 3D electron microscopy imaging, including widefield montaging TEM, TEM tomography as well as Focused Ion Beam Scanning Electron Microscopy (FIB/SEM) and Serial Block Face (SBF/) SEM. The latter two techniques can yield macromolecular insight into the 3D organization of entire cells and tissues, and thus have the potential to truly revolutionize cell biology.
However, while it is now possible to obtain 10k by 10k by 10k voxel data sets (soon 32kx32kx32k), we do not possess the computational capabilities to deal with such terabytes of data, in terms of visualization, feature extraction/segmentation, annotation, and quantitative analysis. I will discuss the challenges these novel imaging approaches pose, describe how we currently deal with such data sets and discuss some emerging solutions that my lab has helped develop in combination with computer scientists at LBL and elsewhere.
We hope that this meeting can lead to further solutions in order to overcome this enormous bottle-neck that is emerging in structural cell biology imaging.