A research team from the Laboratoire de Biogenèse Membranaire (CNRS/University of Bordeaux), in collaboration with the Bordeaux Imaging Center, has recently developed ROOT-ExM, a novel protocol enabling the application of expansion microscopy to plant tissues, specifically the primary root of Arabidopsis thaliana. This method overcomes key limitations that had so far prevented the use of expansion microscopy in plant systems.

Zoom in on expansion microscopy

Expansion microscopy (ExM) is a super-resolution imaging technique that relies on the physical enlargement of biological specimens embedded in a swellable hydrogel. By expanding the gel, the distance between fluorescent labels increases, thereby enabling nanoscale resolution with conventional microscopes.

©Donatelle Liens

The challenge of plant tissue rigidity

Plant cells are embedded in a dense network of polymers (cell walls) that provide mechanical strength and prevent expansion. These structural barriers are especially problematic for expansion microscopy, as they hinder both the penetration of labeling molecules and the isotropic expansion of the sample.

ROOT-ExM: Tailoring expansion microscopy for plants

To adapt ExM to plant roots, the team developed a two-step strategy combining:

  • A mild cell wall digestion, sufficient to relax the cell wall structure and facilitate labeling,
  • And a plant-optimized ExM protocol, compatible with common fluorescent markers and preserving tissue architecture during expansion.

Promising results

  • ROOT-ExM achieves an approximately 4-fold linear expansion of Arabidopsis roots with minimal deformation.
B) Confocal microscopy images of the same root with the same field of view before expansion and after ROOT-ExM.
– In yellow: Endoplasmic Reticulum
– In blue: Cell wall
C) Expansion factor quantification. The diameter of nuclei was measured before and after expansion.
  • When combined with confocal microscopy, this technique enables nanoscale visualization of intracellular (nuclei, Golgi apparatus) and intercellular (plasmodesmata, microtubules) structures with resolution comparable to advanced super-resolution methods.
Representative images of acquisitions in confocal on nonexpanded samples (left), super-resolution lifetime-STED on nonexpanded samples (middle) and confocal after ROOT-ExM (right). Labelling of Golgi apparatus.
  • In addition, coupling ROOT-ExM with lattice light-sheet microscopy (LLSM) allows 3D reconstructions of cellular processes at nanometric resolution and across large tissue volumes.
Volume acquisition of a root cell labeled with anti-KNOLLE (membrane marker during cell division) after ROOT-ExM and imaged by LLSM.

Looking ahead

ROOT-ExM demonstrates that super-resolution imaging of plant tissues is possible using conventional microscopes and accessible labels. While currently limited to the primary root of Arabidopsis thaliana, this protocol lays the groundwork for expanding ExM to more complex plant organs and other species. Rather than replacing high-end super-resolution techniques, ROOT-ExM stands as a complementary approach, providing an accessible, scalable alternative for plant imaging at the nanoscale.