A practical guide to optimization in X10 expansion microscopy

article A practical guide to optimization in X10 expansion microscopy published yes Sven M Truckenbrodt author 45812BD4-F248-11E8-B48F-1D18A9856A87 Christoph M Sommer author 4DF26D8C-F248-11E8-B48F-1D18A9856A870000-0003-1216-9105 Silvio O Rizzoli author Johann G Danzl author 42EFD3B6-F248-11E8-B48F-1D18A9856A870000-0001-8559-3973 JoDa department Bio department ISTplus - Postdoctoral Fellowships project Optical control of synaptic function via adhesion molecules project Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60–80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis. https://research-explorer.ista.ac.at/download/6052/9619/181031_Truckenbrodt_ExM_NatProtoc.docx application/vnd.openxmlformats-officedocument.wordprocessingml.documentno Nature Publishing Group2019 eng Nature Protocols 30778205 00045989070000810.1038/s41596-018-0117-3 143832–863 Truckenbrodt SM, Sommer CM, Rizzoli SO, Danzl JG. 2019. A practical guide to optimization in X10 expansion microscopy. Nature Protocols. 14(3), 832–863. S. M. Truckenbrodt, C. M. Sommer, S. O. Rizzoli, and J. G. Danzl, “A practical guide to optimization in X10 expansion microscopy,” Nature Protocols, vol. 14, no. 3. Nature Publishing Group, pp. 832–863, 2019. S.M. Truckenbrodt, C.M. Sommer, S.O. Rizzoli, J.G. Danzl, Nature Protocols 14 (2019) 832–863. Truckenbrodt, Sven M., et al. “A Practical Guide to Optimization in X10 Expansion Microscopy.” Nature Protocols, vol. 14, no. 3, Nature Publishing Group, 2019, pp. 832–863, doi:<a href="https://doi.org/10.1038/s41596-018-0117-3">10.1038/s41596-018-0117-3</a>. Truckenbrodt, S. M., Sommer, C. M., Rizzoli, S. O., & Danzl, J. G. (2019). A practical guide to optimization in X10 expansion microscopy. Nature Protocols. Nature Publishing Group. <a href="https://doi.org/10.1038/s41596-018-0117-3">https://doi.org/10.1038/s41596-018-0117-3</a> Truckenbrodt, Sven M, Christoph M Sommer, Silvio O Rizzoli, and Johann G Danzl. “A Practical Guide to Optimization in X10 Expansion Microscopy.” Nature Protocols. Nature Publishing Group, 2019. <a href="https://doi.org/10.1038/s41596-018-0117-3">https://doi.org/10.1038/s41596-018-0117-3</a>. Truckenbrodt SM, Sommer CM, Rizzoli SO, Danzl JG. A practical guide to optimization in X10 expansion microscopy. Nature Protocols. 2019;14(3):832–863. doi:<a href="https://doi.org/10.1038/s41596-018-0117-3">10.1038/s41596-018-0117-3</a> 60522019-02-24T22:59:20Z2025-04-14T07:44:00Z