Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy
Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy
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DOI：
10.3791/54983
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发表时间：
2017-02-01
影响因子：
1.2
通讯作者：
Chung, Roger S.
中科院分区：
文献类型：
--
作者：
Morsch, Marco;Radford, Rowan A. W.;Chung, Roger S.
使用标准的共聚焦设置，可以利用UV消融方法来选择性地诱导细胞损伤，并实时可视化CNS中的单细胞反应和细胞-细胞相互作用。此前，研究损伤后的这些细胞特异性反应通常需要复杂的设置或将细胞或动物转移到不同的非生理环境中，从而混淆了即时和短期分析。例如，药物介导的消融方法通常缺乏研究单细胞反应和直接细胞-细胞相互作用所需的特异性。类似地，虽然高功率脉冲激光消融方法提供了非常好的控制和组织穿透，但它们需要专门的设备，这可能会使细胞反应的实时可视化复杂化。这里描述的改进的UV激光烧蚀方法允许研究人员使用配备405 nm激光的传统共聚焦显微镜以剂量和时间依赖的方式对单个细胞施加压力或杀死单个细胞。该方法被应用于选择性地消融斑马鱼脊髓中周围细胞密集网络内的单个神经元。这种方法揭示了消融神经元的剂量依赖性反应，导致细胞体的碎片和顺向变性沿着轴突在几分钟至几小时内。这种方法使研究人员能够研究单个垂死细胞的命运，重要的是，研究消融部位周围细胞（如小胶质细胞和星形胶质细胞）的即时反应。
Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.