Understanding gene regulation networks in multicellular organisms is crucial to decipher many complex physiological processes ranging from development to aging. One technique to characterize gene expression with tissue-specificity in whole organisms is single-molecule fluorescence in situ hybridization (smFISH). However, this protocol requires lengthy incubation times, and it is challenging to achieve multiplexed smFISH in a whole organism. Multiplexing techniques can yield transcriptome-level information, but they require sequential probing of different genes. The inefficient macromolecule exchange through diffusion-dominant transport across dense tissues is the major bottleneck. In this work, we address this challenge by developing a microfluidic/electrokinetic hybrid platform to enable multicycle smFISH in an intact model organism, Caenorhabditis elegans. We integrate an ion concentration polarization based ion pump with a microfluidic array to rapidly deliver and remove gene-specific probes and stripping reagents on demand in individual animals. Using our platform, we can achieve rapid smFISH, an order of magnitude faster than traditional smFISH protocols. We also demonstrate the capability to perform multicycle smFISH on the same individual samples, which is impossible to do off-chip. Our method hence provides a powerful tool to study individual-specific, spatially resolvable, and large-scale gene expression in whole organisms.

中文翻译：

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通过微流控离子浓差极化实现快速、多周期 smFISH，用于对整个秀丽隐杆线虫中组织特异性基因表达进行原位分析。

了解多细胞生物中的基因调控网络对于破译从发育到衰老的许多复杂的生理过程至关重要。单分子荧光原位杂交 (smFISH) 是一种利用整个生物体的组织特异性来表征基因表达的技术。然而，该方案需要较长的孵育时间，并且在整个生物体中实现多重 smFISH 具有挑战性。多重技术可以产生转录组水平的信息，但它们需要对不同基因进行连续探测。通过致密组织的扩散主导运输进行的低效大分子交换是主要瓶颈。在这项工作中，我们通过开发微流体/动电混合平台来应对这一挑战，以在完整的模型生物体——秀丽隐杆线虫中实现多周期 smFISH。我们将基于离子浓差极化的离子泵与微流体阵列集成在一起，以根据个体动物的需要快速输送和去除基因特异性探针和剥离试剂。使用我们的平台，我们可以实现快速 smFISH，比传统 smFISH 协议快一个数量级。我们还展示了对同一单个样本执行多周期 smFISH 的能力，这是在片外不可能实现的。因此，我们的方法提供了一个强大的工具来研究整个生物体中个体特异性、空间可解析和大规模的基因表达。