Lee Hyewon, Kim Shin Ae, Coakley Sean, Mugno Paula, Hammarlund Marc, Hilliard Massimo A, Lu Hang
School of Chemical & Biomolecular Engineering, Georgia Institute of Technology.
Queensland Brain Institute, The University of Queensland, Brisbane QLD 4072, Australia.
Lab Chip. 2014 Dec 7;14(23):4513-4522. doi: 10.1039/c4lc00789a. Epub 2014 Sep 26.
Selective cell ablation can be used to identify neuronal functions in multicellular model organisms such as Caenorhabditis elegans. The optogenetic tool KillerRed facilitates selective ablation by enabling light-activated damage of cell or subcellular components in a temporally and spatially precise manner. However, the use of KillerRed requires stimulating (5 min-1 h), culturing (~24 h) and imaging (often repeatedly) a large number of individual animals. Current manual manipulation methods are limited by their time-consuming, labor-intensive nature, and their usage of anesthetics. To facilitate large-scale selective ablation, culturing, and repetitive imaging, we developed a densely-packed multi-channel device and used it to perform high-throughput neuronal ablation on KillerRed-expressing animals. The ability to load worms in identical locations with high loading efficiency allows us to ablate selected neurons in multiple worms simultaneously. Our device also enables continuous observation of animals for 24 h following KillerRed activation, and allows the animals to be recovered for behavioural assays. We expect this multi-channel device to facilitate a broad range of long-term imaging and selective illumination experiments in neuroscience.
选择性细胞消融可用于识别多细胞模式生物(如秀丽隐杆线虫)中的神经元功能。光遗传学工具KillerRed能够以时空精确的方式实现细胞或亚细胞成分的光激活损伤,从而促进选择性消融。然而,使用KillerRed需要对大量个体动物进行刺激(5分钟至1小时)、培养(约24小时)和成像(通常需要反复进行)。当前的手动操作方法受限于其耗时、劳动强度大的特点以及麻醉剂的使用。为了便于大规模的选择性消融、培养和重复成像,我们开发了一种密集排列的多通道装置,并使用它对表达KillerRed的动物进行高通量神经元消融。以高加载效率将线虫加载到相同位置的能力使我们能够同时消融多条线虫中的选定神经元。我们的装置还能够在KillerRed激活后对动物进行24小时的连续观察,并允许动物恢复以进行行为分析。我们期望这种多通道装置能够促进神经科学领域广泛的长期成像和选择性照明实验。