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测量复杂的原位基因表达， 基于三项创新。首先，我们将开发一个大的调色板光谱条形码 荧光聚合物点（Pdot）探针，其将用于标记完整组织内的mRNA; Pdot的独特光谱特征允许鉴定多达数千种不同的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， 并基于可溶胀的聚合物水凝胶来扩张组织。这些改进的材料将降低mRNA 因此，可以在没有空间重叠的情况下一次读出许多浓度。三是 建议开发一种新的光片显微镜，能够快速询问组织体积， 读出各个Pdot探针上的光谱条形码。我们将一起使用这些工具来研究 小鼠视觉皮层的大脑发育，哺乳动物大脑的模型系统。我们 该方法还应广泛适用于其他复杂组织和/或小细胞的研究。 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.