ExPOSE: a comprehensive toolkit to perform expansion microscopy in plant protoplast systems

Expansion microscopy (ExM) achieves nanoscale imaging by physical expansion of fixed biological tissues embedded in a swellable hydrogel, enhancing the resolution of any optical microscope several-fold. While ExM is commonly used in animal cells and tissues, there are few plant-specific protocols. Protoplasts are a widely used cell system across plant species, especially in studying biomolecule localization. Here, we present an approach to achieve robust expansion of plant protoplasts, termed Expansion microscopy in plant PrOtoplast SystEms (ExPOSE). We demonstrate that coupling ExPOSE with other imaging techniques, immunofluorescence and in situ hybridization chain reaction to visualize proteins and mRNAs, respectively, greatly enhances the spatial resolution of endogenous biomolecules. Additionally, in this study, we tested the effectiveness and versatility of this technique to observe biomolecular condensates in Arabidopsis protoplasts and transcription factors in maize protoplasts at increased resolution. ExPOSE can be relatively inexpensive, fast, and simple to implement.