Recent development in fluorescence-based molecular tools has contributed significantly to developmental studies, including embryogenesis. Many of these tools rely on multiple steps of sample manipulation, so obtaining large sample sizes presents a major challenge as it can be labor-intensive and time-consuming. However, large sample sizes are required to uncover critical aspects of embryogenesis, for example, subtle phenotypic differences or gene expression dynamics. This problem is particularly relevant for single-molecule fluorescence in situ hybridization (smFISH) studies in Caenorhabditis elegans embryogenesis. Microfluidics can help address this issue by allowing a large number of samples and parallelization of experiments. However, performing efficient reagent exchange on chip for large numbers of embryos remains a bottleneck. Here, we present a microfluidic pipeline for large-scale smFISH imaging of C. elegans embryos with minimized labor. We designed embryo traps and engineered a protocol allowing for efficient chemical exchange for hundreds of C. elegans embryos simultaneously. Furthermore, the device design and small footprint optimize imaging throughput by facilitating spatial registration and enabling minimal user input. We conducted the smFISH protocol on chip and demonstrated that image quality is preserved. With one device replacing the equivalent of 10 glass slides of embryos mounted manually, our microfluidic approach greatly increases throughput. Finally, to highlight the capability of our platform to perform longitudinal studies with high temporal resolution, we conducted a temporal analysis of par-1 gene expression in early C. elegans embryos. The method demonstrated here paves the way for systematic high-temporal-resolution studies that will benefit large-scale RNAi and drug screens and in systems beyond C. elegans embryos.

中文翻译：

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用于秀丽隐杆线虫胚胎基因表达研究的高时间分辨率 smFISH 方法

基于荧光的分子工具的最新发展对包括胚胎发生在内的发育研究做出了重大贡献。其中许多工具依赖于样本操作的多个步骤，因此获得大样本量是一项重大挑战，因为它可能是劳动密集型且耗时的。然而，需要大样本量才能揭示胚胎发生的关键方面，例如细微的表型差异或基因表达动态。这个问题与秀丽隐杆线虫胚胎发生的单分子荧光原位杂交（smFISH）研究特别相关。微流体可以通过允许大量样本和并行实验来帮助解决这个问题。然而，在芯片上对大量胚胎进行有效的试剂交换仍然是一个瓶颈。在这里，我们提出了一种微流体管道，用于以最少的劳动力对线虫胚胎进行大规模 smFISH 成像。我们设计了胚胎陷阱并设计了一个方案，允许同时对数百个秀丽隐杆线虫胚胎进行有效的化学交换。此外，设备设计和小占地面积通过促进空间配准和最小化用户输入来优化成像吞吐量。我们在芯片上进行了 smFISH 协议，并证明图像质量得以保留。我们的微流体方法用一台设备取代了相当于 10 张手动安装的胚胎载玻片，大大提高了通量。最后，为了强调我们的平台以高时间分辨率进行纵向研究的能力，我们对早期秀丽隐杆线虫胚胎中的par-1基因表达进行了时间分析。这里展示的方法为系统性高时间分辨率研究铺平了道路，这将有利于大规模 RNAi 和药物筛选以及线虫胚胎以外的系统。