A Multifunctional Anchor for Multimodal Expansion Microscopy

Abstract

Abstract In situ imaging of biomolecular location with nanoscale resolution enables mapping of the building blocks of life throughout biological systems in normal and disease states. Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to ExM is the equipping of different kinds of molecule, with different kinds of anchoring moiety, so they can all be pulled apart by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables multiple kinds of molecules ( e.g., proteins and RNAs) to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and greatly reduces cost (by 2-10 fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this unified ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructure, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues, and can support the visualization of other kinds of biomolecular species as well. Thus, uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.