Super-resolution expansion microscopy in plant roots

Abstract

Abstract Super-resolution methods provide far better spatial resolution than the optical diffraction limit of about half the wavelength of light (∼200–300 nm). Nevertheless, they have yet to attain widespread use in plants, largely due to plants' challenging optical properties. Expansion microscopy (ExM) improves effective resolution by isotropically increasing the physical distances between sample structures while preserving relative spatial arrangements and clearing the sample. However, its application to plants has been hindered by the rigid, mechanically cohesive structure of plant tissues. Here, we report on whole-mount ExM of thale cress (Arabidopsis thaliana) root tissues (PlantEx), achieving a 4-fold resolution increase over conventional microscopy. Our results highlight the microtubule cytoskeleton organization and interaction between molecularly defined cellular constituents. Combining PlantEx with stimulated emission depletion microscopy, we increase nanoscale resolution and visualize the complex organization of subcellular organelles from intact tissues by example of the densely packed COPI-coated vesicles associated with the Golgi apparatus and put these into a cellular structural context. Our results show that ExM can be applied to increase effective imaging resolution in Arabidopsis root specimens.

Code and data availability

The paper's PlantEx expansion microscopy imaging data are deposited in ISTA's public repository, and the authors' custom analysis code (including the BigWarp-based expansion-factor script) is publicly available on GitHub. The Click-ExM repository is cited prior work whose method was adapted, not a paper-specific asset.