Electron tomography (ET) is one of the two EM methods to obtain three- dimensional ultra-structural images. It relies on tilting the specimen and the automated acquisition of multiple tilt projections. It overcomes one of the major limitations of thin sections TEM that is restricted to 2D-images.
Study of Shigella flexneri infection of Hela cells by 3D Correlative Light-Electron Tomography: in order to identify and image the subsequent entry steps of Shigella into host cells, we developed an innovative methodology that combines correlative light and transmission electron microscopy with serial sectioning and tomography on identical samples. The final aim is to couple the functional information derived by 3D confocal microscopy with the high-resolution structural information from 3D electron tomography. Bacteria entry events identified by actin foci were imaged in 3D by confocal microscopy (fig.A, boxed area: bacteria are labelled in red and GFP-actin in green), processed for transmission electron microscopy and the same entry event were then imaged in 3D by electron tomography (fig.B). The reconstructed tomographic volume was then modelled in 3D (fig.C & D), highlighting novel vesicular structures that are docking or fusing with the phagocytic vacuole. (Scale bars: 1 µm)