Expansion microscopy of C. elegans
Expansion microscopy of C. elegans
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DOI：
10.7554/elife.46249
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发表时间：
2020-05-01
期刊：
影响因子：
7.7
通讯作者：
Boyden, Edward S.
中科院分区：
文献类型：
--
作者：
Yu, Chih-Chieh (Jay);Barry, Nicholas C.;Boyden, Edward S.
我们最近开发了膨胀显微镜（ExM），通过化学处理的水凝胶包埋组织的各向同性膨胀，以类似于70 nm的分辨率（类似于4.5倍线性膨胀）实现样品的纳米级精确成像。C的ExM。秀丽隐杆线虫受到其角质层的挑战，角质层坚硬，抗体无法穿透。在这里，我们提出了一个策略，扩展C。elegans（ExCel），以扩展固定的、完整的C.优雅ExCel能够以类似于65-75 nm的分辨率（3.3-3.8x线性扩展）同时读取荧光蛋白、RNA、DNA位置和解剖结构。我们还开发了保留表位的ExCel，它能够对抗体染色的内源性蛋白质进行成像，以及迭代的ExCel，它能够在20倍线性扩增后对荧光蛋白进行成像。我们展示了用于映射突触蛋白的ExCel工具箱的实用性，用于识别先前未报道的细胞连接处的蛋白质，以及用于同一动物的多个单独神经元中的基因表达分析。
We recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at similar to 70 nm resolution (with similar to 4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of similar to 65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal.