A practical guide to optimization in X10 expansion microscopy

2019 | journal article; research paper. A publication with affiliation to the University of Göttingen.

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​A practical guide to optimization in X10 expansion microscopy​
Truckenbrodt, S.; Sommer, C.; Rizzoli, S. O.   & Danzl, J. G.​ (2019) 
Nature Protocols14(3) pp. 832​-863​.​ DOI: https://doi.org/10.1038/s41596-018-0117-3 

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Authors
Truckenbrodt, Sven; Sommer, Christoph; Rizzoli, Silvio O. ; Danzl, Johann G.
Abstract
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.
Issue Date
2019
Journal
Nature Protocols 
Project
info:eu-repo/grantAgreement/EC/FP7/614765/EU//NEUROMOLANATOMY
SFB 1286: Quantitative Synaptologie 
SFB 1286 | Z03: Unkomplizierte multispektrale, superauflösende Bildgebung durch zehnfache Expansionsmikroskopie 
Working Group
RG Rizzoli (Quantitative Synaptology in Space and Time) 
ISSN
1750-2799
Language
English

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