Tomography / Cryo-FIB SEM

Electron tomography allows a protein or cell sample to be visualised in three dimensions from two-dimensional images obtained by imaging the object of interest in different orientations: 40 to 60 images of the same sample are collected, after tilting it at different angles to the electron beam. After alignment of the series of tilted images, a tomogram of the sample is obtained. Electron tomography can be performed at room temperature or under cryogenic conditions. It can also be coupled with ultra-(cryo)-microtomy to image cell sections.

At room temperature, the three-dimensional information allows, for example, the visualisation of a gold bead coupled to an antibody, thus allowing the localisation of an antigen in the volume of a cell or a cell section.

<multi>[fr]Un échantillon présent dans la glace vitreuse est imagé à différents angles par rapport au faisceau d'électrons. À partir de cette série d'images inclinées, un cryo-tomogramme ou volume 3D de l'échantillon est reconstruit. Plusieurs sous-volumes contenant des sous-domaines identiques de l'échantillon sont extraits, alignés et moyennés pour obtenir un modèle 3D. [en]A vitrified sample is imaged at different angles to the electron beam. From this series of tilted images, a cryo-tomogram or 3D volume of the sample is reconstructed. Several sub-volumes containing identical sub-domains of the sample are extracted, aligned and averaged to obtain a 3D model. </multi>
Principle of cryo-electron tomography ((c) D. Levy)

Cryo-electron tomography makes it possible to analyse multi-component protein complexes or viruses that are difficult to purify outside their native environment and to obtain their 3D organisation. Compared to "classical" cryo-microscopy (single particles), the resolution is only a few nm, the signal-to-noise ratio being lower (lower electron dose per image and thicker sample (100-300 nm)).
By averaging the sub-tomograms, it is possible to obtain 3D models of objects present in the tomograms (the particles are in this case sub-volumes and not 2D projections). The alignment and averaging of a large number of sub-volumes will increase the noise ratio and therefore the resolution.

Cellular cryo-electron tomography is a state-of-the-art technique that is rapidly evolving towards high-resolution in situ imaging. This technique can be performed on small prokaryotes, at the periphery of adherent eukaryotic cells or on isolated organelles. For larger samples, cryo-tomography can also be used on cryogenic sections of cells, obtained by CEMOVIS or cryo-FIB SEM.