Spatially Resolved Transcriptomics Enabled by Ultrabright Pdot Probes for Interrogation of Complex Tissues

摘要 我们建议开发一种变革性的方法来原位测量复合体中的基因表达。 基于三项创新的纸巾。首先，我们将开发一个大型调色板--光谱条形码 荧光聚合点(PDot)探针，用于标记完整组织内的mRNA； Pdots的独特光谱特征允许识别多达数千个不同的mRNA 分子作为基因表达的读数。第二，我们建议开发改进的材料，以清除 以及基于可膨胀聚合物水凝胶的组织扩张。这些改良材料将降低信使核糖核酸 浓度，以便可以一次读出多个，而不会在空间上重叠。第三，我们 建议开发一种新的光片显微镜，它能够快速询问组织体积和 读出各个PDOT探头上的光谱条形码。我们将一起使用这些工具来研究 小鼠视觉皮质的大脑发育，哺乳动物大脑的一个模型系统。我们的 方法学也应广泛适用于其他复杂组织和或小组织的研究 有机体。

Abstract We propose to develop a transformative approach to measure gene expression in situ in complex tissues based on three innovations. First, we will develop a large palette spectrally-barcoded fluorescent polymer-dot (Pdot) probes which will be used to label mRNA within an intact tissue; the unique spectral signatures of the Pdots allow the identification of up to thousands of distinct mRNA molecules as a readout of gene expression. Second, we propose to develop improved materials to clear and expand tissues based on swellable polymer hydrogels. These improved materials will lower mRNA concentrations so that many can be read out at one time without spatially overlapping. Third, we propose to develop a new light sheet microscope which is able to rapidly interrogate tissue volumes and read out the spectral barcodes on the individual Pdot probes. We will use these tools together to study brain development of the mouse visual cortex, a model system for the mammalian brain. Our methodology should also be widely applicable to the study of other complex tissues and or small organisms.