Visualizing the Molecular Socialogy at the HeLa Nucleus in Situ

Abstract

Cellular functions arise from an intricate network of macromolecular interactions. Hence it is of fundamental importance to be able to decipher the ‘molecular sociology’ of cells, ideally by direct visualization - the goal of visual proteomics. Traditional electron microscopy has established cellular ultrastructure as we know it, but falls short of revealing the molecular landscape of cells. Cryo-electron tomography (ET) provides three-dimensional images of unperturbed cellular environments at increasingly higher resolutions. Hitherto there were a number of limitations which can now be overcome: Only peripheral thin regions of cells were accessible but thicker regions including nuclear volumes, remained inaccessible; their thickness rendered them non transparent. Focused ion beam micromachining (FIB) changed this and virtually opened ‘windows’ into frozen-hydrated cells. Direct detectors improve the quality of the tomograms substantially and the recently developed Volta phase plate improves (phase) contrast especially for lower spatial frequencies without the need of defocusing. In-focus phase contrast avoids the need of correcting for the contrast transfer function and renders the tomograms directly interpretable. Here we demonstrate that these developments can be used in a synergistic manner to produce 3D images of HeLa cells in situ of unprecedented quality, allowing for direct visualization of macromolecular complexes and their spatial coordination in unperturbed eukaryotic cellular environments.