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首页> 外文期刊>Optics & Laser Technology >Clear(T) immersion optical clearing method for intact 3D spheroids imaging through confocal laser scanning microscopy
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Clear(T) immersion optical clearing method for intact 3D spheroids imaging through confocal laser scanning microscopy

机译：清除（T）通过共聚焦激光扫描显微镜进行完整的3D球体成像的完整3D球状物的浸没光学清除方法

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摘要

Spheroids are 3D in vitro platforms that fill the gap between the 2D cell cultures and animal models on the therapeutics development pipeline. Yet, the methods and equipment used in the in vitro assays are optimized for the analysis of cells cultured as monolayers. For instance, confocal laser scanning microscopy (CLSM) does not allow the observation of thick intact spheroids due to light penetration issues. To overcome this limitation, spheroids treatment with clearing agents started to be explored. Herein, we demonstrate for the first time the application of Clear(T) clearing method for the imaging of propidium iodide (PI) stained spheroids by CLSM. The results demonstrate that the ClearT is a reversible clearing method that does not influence the structure of the spheroid and significantly improved the PI signal penetration depth in about 43%. Additionally, ClearT also enhanced the cells imaging within the spheroid by increasing the cross -penetration depth in 46.6% at 100 mu m of depth. Overall, the results show that ClearT method may allow the improvement of the CLSM accuracy on the evaluation of the cellular death within spheroids prompted by therapeutics. (C) 2018 Elsevier Ltd. All rights reserved.
机译：球状体是3D体外平台，填补了治疗型发育管道的2D细胞培养物和动物模型之间的差距。然而，在体外测定中使用的方法和设备针对作为单层培养的细胞的分析进行了优化。例如，共聚焦激光扫描显微镜（CLSM）由于光穿透问题，不允许观察厚的完整球体。为了克服这种限制，迄今探讨了用清除剂处理的球形处理。在此，我们首次证明了CLSM通过CLSM染色碘化丙酰染色的球状体成像的清除（T）清除方法的应用。结果表明，清晰的是一种可逆的清除方法，不会影响球状体的结构，并且显着改善了PI信号穿透深度约为43％。另外，通过在100μm深度下增加交叉平衡深度，清除也通过增加了46.6％的交叉封闭深度来增强了球状体内的细胞。总体而言，结果表明，清除方法可以允许改善CLSM精度，以提高由治疗剂提示的球状体内的细胞死亡的评估。 （c）2018年elestvier有限公司保留所有权利。

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