TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy.
TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy.
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DOI：
10.1016/j.crmeth.2022.100311
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发表时间：
2022-10-24
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超分辨率显微镜揭示了生物结构的分子组织，直到纳米级。虽然它允许在单细胞，小生物体或薄组织切片中研究蛋白质复合物，但目前还没有与整个脊椎动物胚胎相容的超微结构分析的通用方法。在这里，我们提出了组织超微结构扩展显微镜（TissUExM），一种方法来扩大毫米级和机械异质性的整个胚胎组织，包括果蝇翅盘，整个斑马鱼和小鼠胚胎。TissUExM用于观察内源性蛋白质。它允许定量表征的蛋白质复合物在各种细胞器中的超分辨率在一个范围内的0.3毫米大小的组织使用传统的显微镜。我们通过研究组织特异性纤毛结构异质性和纤毛蛋白过表达后观察到的超微结构缺陷来证明其强度。总的来说，TissUExM是在整个胚胎中进行超微结构研究和原位分子定位的理想选择。我们开发了TissUExM用于机械异质组织的扩增显微镜TissUExM依赖于扩增后标记来定位内源性蛋白质TissUExM有助于在整个胚胎中原位进行定量超微结构分析TissUExM可以应用于整个斑马鱼和小鼠胚胎或飞行翅膀超分辨率显微镜在整个胚胎中具有挑战性，这限制了发育过程中的结构-功能分析。我们开发了一种方法，允许在机械异质毫米级胚胎的定量超微结构分析，优化后的扩展标记，并在高密度环境中的内源性蛋白质的本地化。超分辨率显微镜在整个胚胎中具有挑战性，限制了发育过程中的结构-功能分析。Steib等人开发TissUExM以物理方式扩展异质组织并标记内源性蛋白质复合物。这种方法可以应用于苍蝇翅膀或整个斑马鱼和小鼠胚胎，使定量超微结构分析在纳米尺度上。
Super-resolution microscopy reveals the molecular organization of biological structures down to the nanoscale. While it allows the study of protein complexes in single cells, small organisms, or thin tissue sections, there is currently no versatile approach for ultrastructural analysis compatible with whole vertebrate embryos. Here, we present tissue ultrastructure expansion microscopy (TissUExM), a method to expand millimeter-scale and mechanically heterogeneous whole embryonic tissues, including Drosophila wing discs, whole zebrafish, and mouse embryos. TissUExM is designed for the observation of endogenous proteins. It permits quantitative characterization of protein complexes in various organelles at super-resolution in a range of ∼3 mm-sized tissues using conventional microscopes. We demonstrate its strength by investigating tissue-specific ciliary architecture heterogeneity and ultrastructural defects observed upon ciliary protein overexpression. Overall, TissUExM is ideal for performing ultrastructural studies and molecular mapping in situ in whole embryos. We developed TissUExM for expansion microscopy of mechanically heterogeneous tissues TissUExM relies on post-expansion labeling for localization of endogenous proteins TissUExM facilitates quantitative ultrastructural analysis in situ in whole embryos TissUExM can be applied to whole zebrafish and mouse embryos or to fly wings Super-resolution microscopy is challenging in whole embryos, which limits structure-function analysis during developmental processes. We developed a method allowing quantitative ultrastructural analysis in mechanically heterogeneous millimeter-scaled embryos, optimized for post-expansion labeling, and the localization of endogenous proteins in high-density environments. Super-resolution microscopy is challenging in whole embryos, limiting structure-function analysis during developmental processes. Steib et al. developed TissUExM to physically expand heterogeneous tissues and label endogenous protein complexes. This approach can be applied to fly wings or to whole zebrafish and mouse embryos, enabling quantitative ultrastructural analysis at the nanometer scale.