Institute of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany.
Center for Biostructural Imaging of Neurodegeneration, University of Göttingen Medical Center, Göttingen, Germany.
Nat Protoc. 2019 Mar;14(3):832-863. doi: 10.1038/s41596-018-0117-3.
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.
扩展显微镜是一种相对较新的超分辨率成像方法，它使用可膨胀水凝胶在细胞培养物或组织切片等生物样本中各向同性地增加荧光团之间的物理距离。经典的凝胶配方可实现4×的扩展因子，分辨率为 60-80nm。我们最近开发了 X10 显微镜，它使用一种凝胶，可实现10×的扩展因子，分辨率为~25nm。在这里，我们提供了 X10 扩展显微镜的分步协议。一个典型的实验包括七个连续的阶段：（i）免疫染色，（ii）固定，（iii）聚合，（iv）均化，（v）扩展，（vi）成像，和（vii）验证。这里提出的方案包括优化建议、陷阱及其解决方案，以及详细的指南，这些应该可以提高重现性。尽管我们的方案侧重于 X10 扩展显微镜，但我们详细说明了这些建议中哪些也适用于经典的 4 倍扩展显微镜。我们使用来自大鼠的原代海马神经元来说明我们的方案，但我们的方法可以用于其他原代细胞或感兴趣的培养细胞系。本方案将使任何具有免疫染色基本经验并可使用荧光显微镜的研究人员都能够进行 X10 超分辨率显微镜检查。该过程需要 3 天，大约需要 5 小时主动处理样本进行标记和扩展，另外还需要大约 3 小时进行成像和分析。
