A fully automated microfluidic femtosecond laser axotomy platform for nerve regeneration studies in C. elegans.
A fully automated microfluidic femtosecond laser axotomy platform for nerve regeneration studies in C. elegans.
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DOI：
10.1371/journal.pone.0113917
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Ben-Yakar A
中科院分区：
文献类型：
--
作者：
Gokce SK;Guo SX;Ghorashian N;Everett WN;Jarrell T;Kottek A;Bovik AC;Ben-Yakar A
飞秒激光纳米手术已被广泛接受为轴突损伤模型，使神经再生研究的小模型生物，秀丽隐杆线虫。为了克服人工蠕虫处理技术的时间限制，必须采用自动化和新的固定技术来提高这些研究的吞吐量。虽然已经开发了新的微流体固定技术，有望减少轴突切断术所需的时间，但需要自动化程序来最大限度地减少所需的人为干预量，并加速对高通量至关重要的轴突切断术过程。在这里，我们报告了一个全自动的微流体平台，用于在活体秀丽隐杆线虫中进行荧光标记神经元的激光轴突切断术。所提出的自动化过程将在单个蠕虫内执行轴切术所需的时间减少到1017秒/蠕虫，比手动方法快至少一个数量级。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实现了完全自动化。微流控装置包括T形结构和三维微流控互连以连续地输送、定位和定位蠕虫。图像处理算法可以识别和精确定位消融靶向的轴突。在自动化系统和麻醉剂手动进行的轴突切断术之间，没有观察到统计学显著差异。自动轴切术的总体成功率为67.4±3.2%（236/350），平均处理速度为17.0±2.4 s。这个全自动化的平台建立了一个有前途的方法，在C神经再生的前瞻性全基因组筛选。elegans在一个真正的高通量的方式。
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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