Femtosecond laser nanosurgery has been widely accepted as an axonal injury model, enabling nerve regeneration studies in the small model organism, Caenorhabditis elegans. To overcome the time limitations of manual worm handling techniques, automation and new immobilization technologies must be adopted to improve throughput in these studies. While new microfluidic immobilization techniques have been developed that promise to reduce the time required for axotomies, there is a need for automated procedures to minimize the required amount of human intervention and accelerate the axotomy processes crucial for high-throughput. Here, we report a fully automated microfluidic platform for performing laser axotomies of fluorescently tagged neurons in living Caenorhabditis elegans. The presented automation process reduces the time required to perform axotomies within individual worms to ∼17 s/worm, at least one order of magnitude faster than manual approaches. The full automation is achieved with a unique chip design and an operation sequence that is fully computer controlled and synchronized with efficient and accurate image processing algorithms. The microfluidic device includes a T-shaped architecture and three-dimensional microfluidic interconnects to serially transport, position, and immobilize worms. The image processing algorithms can identify and precisely position axons targeted for ablation. There were no statistically significant differences observed in reconnection probabilities between axotomies carried out with the automated system and those performed manually with anesthetics. The overall success rate of automated axotomies was 67.4±3.2% of the cases (236/350) at an average processing rate of 17.0±2.4 s. This fully automated platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration in C. elegans in a truly high-throughput manner.
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机译:飞秒激光纳米手术已被广泛接受为轴突损伤模型,可以在小型模型秀丽隐杆线虫中进行神经再生研究。为了克服手动蠕虫处理技术的时间限制,在这些研究中必须采用自动化和新的固定技术来提高吞吐量。尽管已开发出有望减少切开术所需时间的新的微流固定技术,但仍需要一种自动化程序来最大程度地减少所需的人工干预并加速对高通量至关重要的切开术过程。在这里,我们报告了一个全自动的微流控平台,用于在秀丽隐杆线虫中进行荧光标记神经元的激光轴切术。提出的自动化过程将在单个蠕虫中进行轴切术所需的时间减少到约17 s /蠕虫,比手动方法至少快一个数量级。通过独特的芯片设计和完全由计算机控制并与高效,准确的图像处理算法同步的操作序列,可以实现完全自动化。微流控设备包括T形架构和三维微流控互连,以连续运输,定位和固定蠕虫。图像处理算法可以识别并精确定位靶向消融的轴突。在使用自动系统进行的静脉切开术和使用麻醉剂手动进行的切开术之间,在重新连接概率上没有观察到统计学上的显着差异。自动切开术的总成功率为案例的67.4±3.2%(236/350),平均处理率为17.0±2.4 s。这个完全自动化的平台为以真正高通量的方式对秀丽隐杆线虫的神经再生进行全基因组前瞻性筛选建立了一种有前途的方法。
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