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

Authors:

Magali S Grison, Guillaume Maucort, Amandine Dumazel, Dorian Champelovier, Yutaro Shimizu, Yohann Boutté, Mónica Fernández-Monreal, Emmanuelle M Bayer

Keywords:

Expansion microscopy; ExM; ROOT-ExM; plant tissue; Arabidopsis thaliana; isotropic expansion; super-resolution microscopy;

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.

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