Visualizing cellular processes at the molecular level by cryo-electron tomography
Visualization of the three-dimensional (3-D) organization of a eukaryotic cell, with its dynamic organelles, cytoskeletal structures, and distinct protein complexes in their native context, requires a non-invasive imaging technique of high resolution combined with a method of arresting cellular elements in their momentary state of function. Vitrification of cells ensures close-to-life preservation of the molecular architecture of actin networks and organelles. With the advent of automated electron tomography it has becomes possible to obtain tomographic data sets of frozen hydrated specimen. By electron tomography 3-D information from large pleomorphous structures, as cell organelles or whole cells can be retrieved with ‘molecular resolution’. At that resolution it becomes possible to detect and identify specific macromolecular complexes on the basis of their structural signature.
Here we employed cryo-electron tomography to eukaryotic cells grown directly on an EM grid. Combining fluorescent microscopy we can navigate within cells and acquire meaningful tomographic reconstructions. Thus, we have analysed unlabelled cellular structures within intact eukaryotic, such as the nuclear periphery and the cell adhesion machinery.