ROOT-ExM: Super-Resolution Imaging of Proteins in Arabidopsis Roots by Expansion Microscopy
Abstract
Conventional light microscopy is limited in resolution by the diffraction limit of light, restricting the visualization of the nanoscale organization of biomolecules. Expansion microscopy (ExM) has emerged as a powerful technique to overcome this barrier by physically expanding the specimen embedded in a swellable hydrogel without requiring specialized or high-cost imaging hardware. ExM is widely used in animal models, whereas its application to plant tissues has been challenging due to their multicellularity, in which each cell is encompassed by the rigid cell wall, which resists the expansion forces and prevents isotropic swelling. Here, we describe a robust and optimized ExM protocol specifically designed for Arabidopsis thaliana root tissues. This protocol details critical steps, including immunostaining, anchoring, gelation, denaturation, cell wall digestion, and expansion. Our method achieves an expansion factor of approximately 4.3×, enabling an effective lateral resolution of ~60 nm using a standard confocal microscope. We demonstrate the visualization of microtubules with preserved ultrastructure. This accessible protocol allows plant researchers to perform super-resolution imaging without specialized optical equipment, facilitating detailed structural analysis of plant cells. Key features • Expansion microscopy to break the diffraction barrier by increasing the physical distances between proteins while preserving relative spatial relationships and fluorescence signals. • 4-fold expansion of Arabidopsis root tissues. • 3D super-resolution imaging. • Deep-tissue imaging thanks to optical clearing associated with expansion of hydrogel-embedded specimens.
Related
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- Dual Color tau-STED Super Resolution Microscopy in Arabidopsis Root Tip
- Labelling strategies and expansion factors in ExM: key steps towards super-resolution microscopy
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BibTeX
@article{shimizu2026rootexm,
title = {{ROOT-ExM: Super-Resolution Imaging of Proteins in Arabidopsis Roots by Expansion Microscopy}},
author = {Yutaro Shimizu and Guillaume Maucort and Mónica Fernández-Monreal and Florentin Dumas and Emmanuelle M Bayer and Yohann Boutté and Magali S Grison},
journal = {Bio-protocol},
year = {2026},
month = jun,
doi = {10.21769/bioprotoc.5707},
url = {https://hal.science/hal-05843886v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - ROOT-ExM: Super-Resolution Imaging of Proteins in Arabidopsis Roots by Expansion Microscopy
AU - Yutaro Shimizu
AU - Guillaume Maucort
AU - Mónica Fernández-Monreal
AU - Florentin Dumas
AU - Emmanuelle M Bayer
AU - Yohann Boutté
AU - Magali S Grison
PY - 2026
DA - 2026/06/05
JO - Bio-protocol
DO - 10.21769/bioprotoc.5707
UR - https://hal.science/hal-05843886v1
AB - Conventional light microscopy is limited in resolution by the diffraction limit of light, restricting the visualization of the nanoscale organization of biomolecules. Expansion microscopy (ExM) has emerged as a powerful technique to overcome this barrier by physically expanding the specimen embedded in a swellable hydrogel without requiring specialized or high-cost imaging hardware. ExM is widely used in animal models, whereas its application to plant tissues has been challenging due to their multicellularity, in which each cell is encompassed by the rigid cell wall, which resists the expansion forces and prevents isotropic swelling. Here, we describe a robust and optimized ExM protocol specifically designed for Arabidopsis thaliana root tissues. This protocol details critical steps, including immunostaining, anchoring, gelation, denaturation, cell wall digestion, and expansion. Our method achieves an expansion factor of approximately 4.3×, enabling an effective lateral resolution of ~60 nm using a standard confocal microscope. We demonstrate the visualization of microtubules with preserved ultrastructure. This accessible protocol allows plant researchers to perform super-resolution imaging without specialized optical equipment, facilitating detailed structural analysis of plant cells. Key features • Expansion microscopy to break the diffraction barrier by increasing the physical distances between proteins while preserving relative spatial relationships and fluorescence signals. • 4-fold expansion of Arabidopsis root tissues. • 3D super-resolution imaging. • Deep-tissue imaging thanks to optical clearing associated with expansion of hydrogel-embedded specimens.
SN - 2331-8325
ER -