Visualizing neurons one-by-one in vivo: Optical dissection and reconstruction of neural networks with reversible fluorescent proteins
Visualizing neurons one-by-one in vivo: Optical dissection and reconstruction of neural networks with reversible fluorescent proteins
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DOI：
10.1002/dvdy.20826
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发表时间：
2006-08-01
影响因子：
2.5
通讯作者：
Hatta, Kohei
中科院分区：
文献类型：
--
作者：
Aramaki, Shinsuke;Hatta, Kohei
大量的轴突和树突混杂成束状，形成突触和肾小球。特别是在实时成像过程中，当单个神经元在空间上连续并以相同的荧光颜色标记时，通常不可能区分它们。为了解决这个问题，我们利用了Dronpa，这是一种绿色荧光蛋白，其荧光可以用强蓝光擦除，并用紫光或紫外光可逆地突出显示。我们首先使用Ga 14-UAS系统可视化了具有荧光Dronpa的神经网络。在轴突导航的延时成像过程中，我们完全消除了Dronpa荧光;从索马顺向或从轴突逆向重新突出显示单个神经元;然后对其他单个神经元重复此过程。在收集了几个单独神经元的图像后，我们将它们重新组合成多种伪彩色以重建网络。我们还成功地使用双光子激发显微镜重新突出显示Dronpa，以标记位于组织内部的单个细胞，并能够展示延伸轴突的Mauthner神经元的可视化。这些“光学解剖”技术有可能在未来实现自动化，并可能提供一种有效的手段，以确定在单细胞水平上的形态发生和网络形成的基因功能。
A great many axons and dendrites intermingle to fasciculate, creating synapses as well as glomeruli. During live imaging in particular, it is often impossible to distinguish between individual neurons when they are contiguous spatially and labeled in the same fluorescent color. In an attempt to solve this problem, we have taken advantage of Dronpa, a green fluorescent protein whose fluorescence can be erased with strong blue light, and reversibly highlighted with violet or ultraviolet light. We first visualized a neural network with fluorescent Dronpa using the Ga14-UAS system. During the time-lapse imaging of axonal navigation, we erased the Dronpa fluorescence entirely; re-highlighted it in a single neuron anterogradely from the soma or retrogradely from the axon; then repeated this procedure for other single neurons. After collecting images of several individual neurons, we then recombined them in multiple pseudo-colors to reconstruct the network. We have also successfully re-highlighted Dronpa using two-photon excitation microscopy to label individual cells located inside of tissues and were able to demonstrate visualization of a Mauthner neuron extending an axon. These "optical dissection" techniques have the potential to be automated in the future and may provide an effective means to identify gene function in morphogenesis and network formation at the single cell level.