Preprint Technical Note Version 1 Preserved in Portico This version is not peer-reviewed

Expansion Microscopy on Saccharomyces cerevisiae

Version 1 : Received: 8 March 2022 / Approved: 10 March 2022 / Online: 10 March 2022 (10:51:02 CET)

How to cite: Korovesi, A.G.; Morgado, L.; Fumasoni, M.; Henriques, R.; Heil, H.; Del Rosario, M. Expansion Microscopy on Saccharomyces cerevisiae. Preprints 2022, 2022030146 (doi: 10.20944/preprints202203.0146.v1). Korovesi, A.G.; Morgado, L.; Fumasoni, M.; Henriques, R.; Heil, H.; Del Rosario, M. Expansion Microscopy on Saccharomyces cerevisiae. Preprints 2022, 2022030146 (doi: 10.20944/preprints202203.0146.v1).

Abstract

The unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross- linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

Keywords

expansion microscopy; yeast; Saccharomyces cerevisiae; super-resolution

Subject

LIFE SCIENCES, Biophysics

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