Fluorescence microscopy provides specific visualisation of biological targets and interactions. Super-resolution fluorescence methods bypass the ~200 nm visible light diffraction limit to reach nanoscale resolutions (10 – 50 nm) suited for subcellular investigations. Conventional super-resolution methods can be performed using specialised optical hardware for deterministic methods such as STED and SIM, or with fluorophore photoswitching for single molecule localization methods such as dSTORM and PAINT [ 1]. These approaches, however, are still bound by the spatial constraints of the dense cellular environment that may limit antibody-based labelling strategies or single molecule localisation precision.
Expansion microscopy (ExM) offers resolution gain through the physical enlargement of the sample, magnifying previously subdiffraction detail into view on diffraction-limited microscopes [ 2]. Besides improving imaging resolution, ExM spatially decrowds and clears biological samples. At the subcellular scale, ExM’s spatial decrowding can be leveraged to overcome the dense nuclear environment.
ExM is performed with a fixed sample embedded in an acrylamide-based hydrogel that expands in water, thus expanding the sample. Importantly, the biomolecule(s) of interest (specific/general proteins, nucleic acids, lipids) must be covalently attached to the hydrogel matrix prior to expansion. A linear expansion factor of 4X is routinely achieved with classic ExM protocols [ 2– 4], engendering a 4-fold improvement in spatial resolution in each dimension and a 64-fold increase in sample volume. The performance of ExM is reliant on hydrogel quality determined by hydrogel chemistry, fluorescence labelling strategy and manual manipulation of the sample. For example, an unequal distribution of monomers into the sample will result in localised hydrogel distortions manifesting as anisotropic expansion of biological structures or shearing of the polymerised hydrogel after expansion ( Fig. 1 ).
Fig. 1
ExM hydrogel shearing across different scales. (A) Macro-scale hydrogel shearing across expanded U2OS nuclei labelled with DAPI. (B) Micro-scale shear across a single U2OS cell labelled for the nucleus (DAPI, blue) and microtubules (alpha-tubulin antibody, green).
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