Aoyagi Yuka, Kawakami Ryosuke, Osanai Hisayuki, Hibi Terumasa, Nemoto Tomomi
Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan; Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan.
Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan; Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Hokkaido, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.
PLoS One. 2015 Jan 29;10(1):e0116280. doi: 10.1371/journal.pone.0116280. eCollection 2015.
Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2'-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.
阐明神经回路功能需要可视化厚脑标本内部区域神经元的精细结构。然而，激光扫描显微镜的组织穿透深度受组织光散射和/或吸收的限制。最近，已提出几种光学透明剂用于固定标本的可视化。然而，它们需要复杂的方案或较长的处理时间。在此，我们报告了2,2'-硫代二乙醇（TDE）溶液作为表达黄色荧光蛋白的固定小鼠脑的光学透明剂的效果。将固定脑浸入TDE溶液中可迅速（对于400μm厚的固定脑切片，在30分钟内）提高其透明度，并增强共聚焦显微镜和双光子显微镜下的穿透深度。此外，我们成功地在固定厚脑切片的深部位置沿单个树突可视化树突棘。这些结果表明，我们提出的使用TDE溶液的方案是一种快速且有用的方法，用于对表达荧光蛋白的固定标本进行光学透明处理。
