Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Elife. 2018 Nov 20;7:e40350. doi: 10.7554/eLife.40350.
The stochastic multicolor labeling method 'Brainbow' is a powerful strategy to label multiple neurons differentially with fluorescent proteins; however, the fluorescence levels provided by the original attempts to use this strategy were inadequate. In the present study, we developed a stochastic multicolor labeling method with enhanced expression levels that uses a tetracycline-operator system (Tetbow). We optimized Tetbow for either plasmid or virus vector-mediated multicolor labeling. When combined with tissue clearing, Tetbow was powerful enough to visualize the three-dimensional architecture of individual neurons. Using Tetbow, we were able to visualize the axonal projection patterns of individual mitral/tufted cells along several millimeters in the mouse olfactory system. We also developed a Tetbow system with chemical tags, in which genetically encoded chemical tags were labeled with synthetic fluorophores. This was useful in expanding the repertoire of the fluorescence labels and the applications of the Tetbow system. Together, these new tools facilitate light-microscopy-based neuronal tracing at both a large scale and a high resolution.
随机多色标记方法“Brainbow”是一种用荧光蛋白对多个神经元进行差异标记的有效策略；然而，最初尝试使用该策略提供的荧光水平并不理想。在本研究中，我们开发了一种具有增强表达水平的随机多色标记方法，该方法使用四环素操纵子系统（Tetbow）。我们优化了 Tetbow 用于质粒或病毒载体介导的多色标记。当与组织透明化结合使用时，Tetbow 足以可视化单个神经元的三维结构。使用 Tetbow，我们能够可视化小鼠嗅觉系统中单个嗅球/丛细胞的轴突投射模式长达数毫米。我们还开发了一种带有化学标签的 Tetbow 系统，其中遗传编码的化学标签被合成荧光团标记。这对于扩展荧光标记的范围和 Tetbow 系统的应用非常有用。这些新工具共同促进了基于显微镜的大规模和高分辨率神经元示踪。
