Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs.
Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs.
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DOI：
10.1523/eneuro.0022-15.2015
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发表时间：
2015-05
期刊：
影响因子：
3.4
通讯作者：
Frankland PW
中科院分区：
文献类型：
--
作者：
Epp JR;Niibori Y;Liz Hsiang HL;Mercaldo V;Deisseroth K;Josselyn SA;Frankland PW
CLARITY是一种用于生物标本光学清除和完整成像的新技术。这种方法于2013年推出，但对一些研究人员来说很难实施。开发、改进和使用高通量全脑细胞分辨率成像技术将有助于我们理解大脑的复杂功能。这些技术对于分析完整的神经元形态、解剖和功能连接以及重复的分子表型至关重要。CLARITY是最近推出的一项技术，它可以生成结构完整、光学透明的组织，无需切片即可进行标记和成像。然而，该技术的效用取决于在组织中的光散射脂质被透明水凝胶基质取代的过程中的几个程序变量。在这里，我们系统地改变了许多因素（包括温度、水凝胶组成和聚合条件），以提供一个优化的、高度可复制的CLARITY程序来清除小鼠大脑。我们发现，对于这些制剂，最佳的组织清除需要电泳（不能单独被动清除），在37°C和55°C的温度组合下持续5天。虽然这个方案是针对大脑进行优化的，但我们也表明它可以用于清除和分析各种器官。使用该方案制备的脑或其他组织适用于共聚焦或单平面照明显微镜的高通量成像。
CLARITY is a novel technique for optical clearance and intact imaging of biological specimens. This method was introduced in 2013 but has been difficult for some researchers to implement. The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology—anatomical and functional—connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy.