Nanoscale imaging of RNA with expansion microscopy.
Nanoscale imaging of RNA with expansion microscopy.
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DOI：
10.1038/nmeth.3899
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发表时间：
2016-08
期刊：
影响因子：
48
通讯作者：
Boyden ES
中科院分区：
文献类型：
--
作者：
Chen F;Wassie AT;Cote AJ;Sinha A;Alon S;Asano S;Daugharthy ER;Chang JB;Marblestone A;Church GM;Raj A;Boyden ES
在完整组织中以纳米级精度对RNA身份和位置进行成像的能力对于在正常和病理生物环境中定义细胞类型和状态具有极大的意义。在这里，我们提出了一种策略的RNA的扩展显微镜（ExM）。我们开发了一种小分子接头，使RNA能够共价连接到整个生物标本中合成的可溶胀的凝胶上。然后，扩增后，RNA的荧光原位杂交（FISH）成像可以在培养的细胞和完整的脑组织中以高产率和特异性、单分子精确度进行。扩增FISH（ExFISH）使RNA去聚集并支持单分子信号的扩增（即，通过杂交链反应（HCR））以及多重RNA FISH读出。因此，ExFISH能够在厚标本中使用衍射限制显微镜对RNA结构和位置进行超分辨率成像，例如完整的脑组织和其他对生物学和医学重要的组织。
The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy (ExM) of RNA. We developed a small molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, post-expansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity, with single molecule precision, in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) de-crowds RNAs and supports amplification of single molecule signals (i.e., via hybridization chain reaction (HCR)) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.