Resolving protein organization in cells with nanometer resolution

Sauer, M.; Eilts, J.; Jungblut, M.; Helmerich, D. A.; Sachs, S.; Werner, C.; Doose, S.; Kollmannsberger, P.

doi:10.1101/2025.08.11.669713

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bioRxiv · 10.1101/2025.08.11.669713

Resolving protein organization in cells with nanometer resolution

Sauer, M.·Eilts, J.·Jungblut, M.·Helmerich, D. A.·Sachs, S.·Werner, C.·Doose, S.·Kollmannsberger, P.

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Abstract

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

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Sauer, M., Eilts, J., Jungblut, M., Helmerich, D. A., Sachs, S., Werner, C., Doose, S., Kollmannsberger, P.. 2025-08-13. Resolving protein organization in cells with nanometer resolution. https://doi.org/10.1101/2025.08.11.669713

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