Root expansion microscopy: A robust method for super resolution imaging in Arabidopsis

Abstract 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 (Arabidopsis thaliana). ROOT-ExM achieves isotropic expansion with a 4-fold increase in resolution, enabling super-resolution microscopy comparable to stimulated emission depletion (STED) microscopy. Labeling is achieved through immunolocalization, compartment-specific dyes, and native fluorescence preservation, while N-hydroxysuccinimide ester-dye conjugates reveal the ultrastructural context of cells alongside specific labeling. We successfully applied ROOT-ExM to image various organelles and subcellular compartments, including the Golgi apparatus, the endoplasmic reticulum, the cytoskeleton, and tiny wall-embedded structures such as plasmodesmata. Combination of ROOT-ExM with STED enabled reaching an unprecedented resolution of plasmodesmata by light microscopy. When combined with lattice light sheet microscopy, ROOT-ExM enabled 3D quantitative analysis of nanoscale cellular processes, such as the size quantification of vesicles near 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 valuable insights into plant subcellular architecture.