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.
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.