RT Journal Article SR Electronic T1 Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx) JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.02.03.428837 DO 10.1101/2021.02.03.428837 A1 Hugo G.J. Damstra A1 Boaz Mohar A1 Mark Eddison A1 Anna Akhmanova A1 Lukas C. Kapitein A1 Paul W. Tillberg YR 2021 UL http://biorxiv.org/content/early/2021/09/15/2021.02.03.428837.abstract AB Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.Competing Interest StatementThe authors have declared no competing interest.