Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics.
Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics.
复制标题
带有微流体学的秀丽隐杆线虫幼虫的长期高分辨率成像。
DOI：
10.1016/j.devcel.2016.11.022
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发表时间：
2017-01-23
影响因子：
11.8
通讯作者：
Siggia ED
中科院分区：
文献类型：
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作者：
Keil W;Kutscher LM;Shaham S;Siggia ED
长期研究表明，C.传统上，秀丽线虫幼虫的发育需要繁琐的人工观察，因为幼虫必须移动以发育，并且现有的固定技术要么干扰发育，要么不适合幼幼虫。在这里，我们提出了一个简单的微流控装置，同时跟踪10 C的发展。线虫幼虫从孵化到成年（~3天）的高时空分辨率。在微室中生长的动物通过压缩定期固定，以允许即使是微弱的荧光信号的高质量成像。利用该装置，我们获得了C.线虫外阴发育，器官发生的范例。我们结合联合收割机Nomarski和多通道荧光显微镜来研究整个胚后发育过程中的细胞命运规范、细胞死亡和转分化。最后，我们通过自动图像配准生成复杂神经分支的延时电影。我们的技术为定量分析C.线虫幼虫发育Keil等人提出了一种微流体装置，能够实现高达10 C的长期、高分辨率、延时显微镜。同时进行。他们收集外阴细胞周期定时统计数据，测量细胞命运规范，转分化和细胞死亡期间荧光转录报告的强度，并在自动注册的z堆栈中可视化复杂的神经突生长。
Long-term studies of C. elegans larval development traditionally require tedious manual observations as larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of 10 C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (~3 days). Animals grown in micro-chambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multi-channel fluorescence microscopy to study processes such as cell-fate specification, cell death, and trans-differentiation throughout postembryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development. Keil et al. present a microfluidics setup, enabling long-term, high-resolution, time-lapse microscopy of up to ten C. elegans larvae simultaneously. They collect vulval cell cycle timing statistics, measure intensities of fluorescent transcriptional reporters during cell fate specification, transdifferentiation and cell death, and visualize complex neurite outgrowth in automatically registered z-stacks.