Abstract
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 10-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.
Highlights
Expansion microscopy (ExM) circumvents the diffraction limit of light microscopy by physically expanding the specimen four-fold in each dimension (Chen et al, 2015; Tillberg et al, 2016)
To systematically explore the expansion recipe space, we developed a streamlined approach for synthesizing dozens of gel recipes and characterizing their mechanical quality in parallel
For every set of gel recipe parameters, we define a recipe family as the set of recipes generated by varying the crosslinker concentration
Summary
Expansion microscopy (ExM) circumvents the diffraction limit of light microscopy by physically expanding the specimen four-fold in each dimension (Chen et al, 2015; Tillberg et al, 2016). This tradeoff of expansion versus gel mechanical integrity has not been explored in a quantitative or systematic way Another gel recipe variant, using a high concentration of the monomer dimethylacrylamide (DMAA), has enough crosslinking through side reactions and polymer chain entanglement that the crosslinker can be omitted entirely, producing ~10-fold expansion in one step (Truckenbrodt et al, 2018). This recipe has been used to expand cultured cells and thin cryosectioned tissue (Truckenbrodt et al, 2019), but reportedly requires rigorous degassing to remove oxygen prior to gelation, making it laborious to use. We show that TREx enables 3D nanoscopic imaging of specific structures stained with antibodies in combination with cellular ultrastructure
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