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.
A better understanding of neuronal network and function requires information about the physiological and transcriptional responses of individual cells within a tissue. At present, there lacks a high-throughput method for interrogating the transcriptome of individual cells of a neuronal network. The proposed technology will address this challenge to provide a tool that can advance our understanding of neuronal networks, both its normal functions as well as its malfunctions in disease states.
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