Séminaire IBS : Visualizing the native cellular organization by coupling cryo-fixation with expansion microscopy (Cryo-ExM)

par Dr Marine Laporte (Institut NeuroMyoGène, Université Claude Bernard, Lyon)

Super-resolution fluorescent microscopy (SRM) allows locating proteins with nanometer resolution in a cellular context, but it requires cell fixation with aldehyde-based chemical crosslinkers, or protein precipitation with cold methanol, which potentially alter the native cellular state and the following interpretations. To bypass this problems, cryo-fixation coupled to SRM has been developed and is now the gold standard for efficient preservation of the native cell ultrastructure. However, this approach is not widely used in fluorescence microscopy owing to implementation. Here, we present a novel, simple approach combining cryo-fixation to ExM (Cryo-ExM), which allows nanoscale observation of cellular compartments in their native state. We demonstrate that Cryo-ExM preserves the native organization of membrane-based organelles such as mitochondria, endoplasmic reticulum, golgi apparatus and lysosomes together with the cytoskeleton components actin and microtubules. Moreover, the possibility to perform multi-labelling allow us to appreciate fine interactions between subcellular compartments such as reticulum and mitochondria or reticulum and microtubules, so far only observed in live SRM. Finally, direct comparison to the gold-standard chemical fixation PFA/GA for the preservation of cellular structure, demonstrates that cryo-fixation bypassed drawbacks associated with this chemical fixation such as antigen accessibility due to strong protein-protein crosslinking. Overall, this simple method provides a basic framework to visualize protein into various subcellular compartments at nanoscale resolution without the chemical fixation artefacts.

Hôte : R. Sadoul (IBS/Groupe Entrée et bourgeonnement des virus à enveloppe)