Protocol for multicolor three-dimensional dSTORM data analysis using MATLAB-based script package Grafeo

International audienceThe plant cell wall, a form of the extracellular matrix, is a complex and dynamicnetwork of polymers mediating a plethora of physiological functions. How poly-saccharides assemble into a coherent and heterogeneous matrix remains mostlyundefined. Further progress requires improved molecular-level visualizationmethods that would gain a deeper understanding of the cell wall nanoarchitec-ture. dSTORM, a type of super-resolution microscopy, permits quantitative nano-imaging of the cell wall. However, due to the lack of single-cell model systems andthe 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-levelmulticolor nanoscopy. Combining dSTORM with spatial statistics, we reveal andcharacterize the ultrastructure of three major polysaccharides, callose, mannan,and cellulose, in the plant cell wall precursor and provide evidence for cellulosestructural re-organization related to callose conten