Resolving endogenous protein organization in cells with nanometer precision

Advances in super-resolution microscopy have outpaced molecular labeling methods, impeding nanometer-scale imaging of endogenous multiprotein complexes in cells. To overcome these limitations, we developed an expansion microscopy (ExM) approach based on double-homogenized hydrogels that enables direct stochastic optical reconstruction microscopy (dSTORM) of 7-8-fold expanded immunolabeled samples. The resulting [~]4-fold increase in effective immunolabeling density resolved the 8 nm distance between neighboring -tubulin molecules in microtubules and the polyhedral lattice in clathrin-coated pits with nanometer precision in cells. Two-color Ex-dSTORM further revealed the molecular organization of RIM scaffolding protein and Munc13-1, an essential synaptic vesicle priming protein, in ring-like structures with diameters of 40-45 nm at the presynapse in hippocampal neurons. Our results demonstrate that Ex-dSTORM resolves the molecular organization of endogenous multiprotein complexes with nanometer spatial resolution in genetically unmodified cells. Thus, it provides a versatile method for the investigation of molecular protein distributions in their physiologically relevant context. One-Sentence SummaryThe optimized combination of dSTORM and double TREx ExM resolves previously unresolvable protein assemblies in cells