Resolving endogenous protein organization in cells with nanometer resolution

Latest refinements in super-resolution microscopy achieved spatial resolutions in the one nanometer range. However, improvement of localization precision advanced faster than labeling methods impeding the translation of such high resolutions to cells. Hence, imaging of the nano-architecture of endogenous multiprotein complexes remains challenging. Here we introduce an expansion microscopy (ExM) method using double-homogenized hydrogels that enables direct stochastic optical reconstruction microscopy (dSTORM) of 7-8-fold expanded immunolabeled samples. The ~4-fold increase in labeling density resolves the 8 nm spacing between neighboring α-tubulin molecules in microtubules, the polyhedral lattice of clathrin-coated pits, and provides evidence for the 8 nm periodicity of α/β-tubulin heterodimers. Two-color Ex-dSTORM further reveals the molecular organization of RIM and the synaptic vesicle priming protein Munc13-1 in 44-48 nm ring-like presynaptic structures in neurons. Ex-dSTORM thus enables nanometer-resolution imaging of endogenous multiprotein complexes in genetically unmodified cells, providing a versatile tool for studying molecular organization in physiological contexts.