The first research publication from the Dey Lab!
We are thrilled to see the final version of our work on yeast ultrastructure expansion microscopy (U-ExM) published and featured on the journal cover! This work was a joint effort and a great collaboration with the Centriole Lab and Loewith Lab at Université de Genève, combining expertise in budding yeast and fission yeast genetics and cell biology with the latest improvements to expansion microscopy. We believe our protocol will be of great interest to many budding and fission yeast labs when faced by questions that demand increased spatial resolution and ultrastructural detail - and we already collaborated with the Cytomotors Lab at EMBL Australia to investigate mitochondria/microtubule interactions!
Briefly, expansion microscopy (ExM) is a method which physically enlarges biological samples by anchoring them within a swellable hydrogel. This allows for an increase in resolution even by conventional microscopy. Ultrastructure ExM (U-ExM) is a variant of the technique which preserves protein epitopes throughout the expansion by using denaturation instead of a protein digestion step. This has the benefit of enabling staining of proteins that are inaccessible under native conditions and mitigates the loss in resolution introduced by bulky antibodies (the linkage error). This technique further allows for a labelling of bulk protein using non-specific dyes which results in images closely resembling electron microscopy images (at lower resolution of course), providing contrast to protein-dense structures and organelles.
We now set out to optimise U-ExM for use in the two model yeasts S. cerevisiae and S. pombe. Sample expansion has previously been hindered by the yeast cell wall, a barrier circumvented by the addition of a digestion step. As a proof of principle, we used U-ExM to quantify the number of nuclear pore complexes (NPCs) across the life cycle of S. pombe. Our results closely match reports in the literature, showing the comparability of U-ExM to other 3D super resolution modalities. On the budding yeast front, Marine and Kerstin investigated the molecular makeup of spindle pole bodies (SPBs), where even in widefield microscopy, protein localisation patterns matched expected measurements.
We did, however, notice some unexpected cellular morphologies – such as non-spherical nuclei – that could not be explained by artefacts of expansion. Changing our mode of fixation from classical, chemical fixation to high pressure freezing (HPF) seems to completely abolish those artefacts. HPF or plunge freezing, techniques predominantly used for electron microscopy, could therefore turn out to be the fixation methods of choice for many other super resolution techniques.
Like any project, this one threw up some unexpected roadblocks that, in hindsight, are rather funny and beautiful to look at:
Our first trial of digesting the cell wall after embedding cells in the gel
Examples of undigested, plunge frozen samples – we had hoped for a destabilisation of the cell wall during the freezing process. Evidently, this was not the case. Batman approves!