Multicolor 3D-dSTORM Reveals Native-State Ultrastructure of Polysaccharides' Network during Plant Cell Wall Assembly

The plant cell wall, a form of the extracellular matrix, is a complex and dynamic network of polymers mediating a plethora of physiological functions. How polysaccharides assemble into a coherent and heterogeneous matrix remains mostly undefined. Further progress requires improved molecular-level visualization methods that would gain a deeper understanding of the cell wall nanoarchitecture. dSTORM, a type of super-resolution microscopy, permits quantitative nanoimaging of the cell wall. However, due to the lack of single-cell model systems and the requirement of tissue-level imaging, its use in plant science is almost absent. Here we overcome these limitations; we compare two methods to achieve three-dimensional dSTORM and identify optimal photoswitching dyes for tissue-level multicolor nanoscopy. Combining dSTORM with spatial statistics, we reveal and characterize the ultrastructure of three major polysaccharides, callose, mannan, and cellulose, in the plant cell wall precursor and provide evidence for cellulose structural re-organization related to callose content.