Many crowded biomolecular structures in cells and tissues are inaccessible to labelling antibodies. To understand how proteins within these structures are arranged with nanoscale precision therefore requires that these structures be decrowded before labelling. Here we show that an iterative variant of expansion microscopy (the permeation of cells and tissues by a swellable hydrogel followed by isotropic hydrogel expansion, to allow for enhanced imaging resolution with ordinary microscopes) enables the imaging of nanostructures in expanded yet otherwise intact tissues at a resolution of about 20 nm. The method, which we named ‘expansion revealing’ and validated with DNA-probe-based super-resolution microscopy, involves gel-anchoring reagents and the embedding, expansion and re-embedding of the sample in homogeneous swellable hydrogels. Expansion revealing enabled us to use confocal microscopy to image the alignment of pre-synaptic calcium channels with post-synaptic scaffolding proteins in intact brain circuits, and to uncover periodic amyloid nanoclusters containing ion-channel proteins in brain tissue from a mouse model of Alzheimer’s disease. Expansion revealing will enable the further discovery of previously unseen nanostructures within cells and tissues.

标记抗体无法接近细胞和组织中许多拥挤的生物分子结构。因此，要了解这些结构中的蛋白质如何以纳米级精度排列，需要在标记之前对这些结构进行去拥挤。在这里，我们展示了膨胀显微镜的迭代变体（细胞和组织被可膨胀的水凝胶渗透，然后是各向同性水凝胶膨胀，以允许使用普通显微镜增强成像分辨率）能够在膨胀但其他方面完整的组织中对纳米结构进行成像分辨率约为 20 nm。我们将这种方法命名为“膨胀揭示”并通过基于 DNA 探针的超分辨率显微镜进行了验证，该方法涉及凝胶锚定试剂以及样品在均质可膨胀水凝胶中的嵌入、膨胀和重新嵌入。膨胀揭示使我们能够使用共聚焦显微镜对完整脑回路中突触前钙通道与突触后支架蛋白的排列进行成像，并从阿尔茨海默病小鼠模型中发现脑组织中含有离子通道蛋白的周期性淀粉样蛋白纳米团簇. 扩展揭示将使进一步发现细胞和组织内以前看不见的纳米结构成为可能。