Li Shuo, Raychaudhuri Sumana, Watanabe Shigeki
Department of Cell Biology, Johns Hopkins School of Medicine; Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health.
Department of Cell Biology, Johns Hopkins School of Medicine.
J Vis Exp. 2017 May 1(123):55664. doi: 10.3791/55664.
Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, "flash-and-freeze," that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.
细胞不断改变其膜结构和蛋白质分布，但要分别在毫秒和纳米级的时间和空间分辨率下观察这些事件极其困难。我们开发了一种时间分辨电子显微镜技术，即“闪光冷冻”，该技术通过光遗传学诱导细胞事件，并通过在刺激后的特定时间点冷冻细胞来观察由此产生的膜动力学。为了演示这项技术，我们在小鼠海马神经元中表达了光敏感阳离子通道视紫红质。一束光刺激神经元活动，并通过突触小泡的融合诱导神经递质从突触末端释放。神经元的光遗传学刺激与高压冷冻相结合，以跟踪突触传递过程中的形态变化。使用商用仪器，我们捕捉到了突触小泡的融合和突触小泡膜的恢复。为了观察事件的顺序，我们生成了大量数据集并进行了盲法分析，因为随着时间的推移，不同细胞中的形态变化是被跟踪的。尽管如此，“闪光冷冻”能够在具有毫秒时间分辨率的电子显微镜图像中观察膜动力学。
