Data from: Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas, from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA, imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (PAssive CLARITY Technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ~20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.

在厚组织样本中对信使RNA（mRNA）分子进行精准且稳定的检测，能够揭示处于原生微环境中的单细胞内的基因表达模式。在获取选定细胞转录组的同时保留空间位置关系，是推动从发育生物学到神经科学等诸多生物学领域发展的关键特性。然而，由于多数组织样本存在较高的自发荧光背景，在不透明的厚样本中稳定检测单分子荧光原位杂交（smFISH）信号颇具挑战。本研究借鉴新兴的动态核酸纳米技术领域的原理，开发出一种基于单分子杂交链式反应（smHCR）的稳定方法，可用于深层组织的多色、多RNA成像。借助该方法，可根据所需成像深度，利用落射荧光显微镜、共聚焦显微镜或选择性平面照明显微镜（SPIM）对单个转录本进行成像。本研究证实，smHCR在细胞培养与完整斑马鱼胚胎的mRNA检测中具有高灵敏度；而将其与SPIM以及PACT（被动透明技术，PAssive CLARITY Technique）组织水凝胶包埋与透明化方法结合后，smHCR可在厚度达0.5毫米的大脑切片深层检测到单个mRNA分子。smHCR可同时实现约20倍的信号放大与衍射极限空间分辨率，为原位检测单个mRNA分子提供了一种稳定且通用的方案，尤其适用于高背景会削弱未扩增smFISH性能的厚组织样本。