Plant Root Expansion Microscopy (ROOT-ExM): A streamlined super resolution method for plants

Expansion microscopy (ExM) has revolutionized biological imaging by physically enlarging samples, surpassing the light diffraction limit and enabling nanoscale visualization using standard microscopes. While extensively employed across a wide range of biological samples, its application to plant tissues is sparse. In this work, we present ROOT-ExM, an expansion method suited for stiff and intricate multicellular plant tissues, focusing on the primary root of Arabidopsis thaliana. ROOT-ExM achieves isotropic expansion with a fourfold increase in resolution, enabling super-resolution microscopy comparable to STimulated Emission Depletion (STED) microscopy. Labelling is achieved through immunolocalization, compartment-specific dyes, and native fluorescence preservation, while N-Hydroxysuccinimide (NHS) ester-dye conjugates reveal the ultrastructural context of cells alongside specific labelling. We successfully applied ROOT-ExM to image various cellular structures, including the Golgi apparatus, the endoplasmic reticulum, the cytoskeleton, and wall-embedded structures such as plasmodesmata. When combined with lattice light sheet microscopy (LLSM), ROOT-ExM achieves 3D quantitative analysis of nanoscale cellular process, revealing increased vesicular fusion in close proximity of the cell plate during cell division. Achieving super-resolution fluorescence imaging in plant biology remains a formidable challenge. Our findings underscore that ROOT-ExM provides a remarkable, cost-effective solution to this challenge, paving the way for unprecedented insights into plant cellular subcellular architecture.