High-resolution 3D imaging of fixed and cleared organoids
High-resolution 3D imaging of fixed and cleared organoids
复制标题
DOI：
10.1038/s41596-019-0160-8
复制
发表时间：
2019-06-01
期刊：
影响因子：
14.8
通讯作者：
Rios, Anne C.
中科院分区：
文献类型：
--
作者：
Dekkers, Johanna F.;Alieva, Maria;Rios, Anne C.
体外3D类器官系统彻底改变了培养皿中器官发育和疾病的建模。荧光显微镜有助于表征类器官的细胞组成，并证明类器官与其原始组织的表型相似性。在这里，我们提供了一个详细的协议，用于执行高分辨率的3D成像的整个类器官窝藏荧光报告和免疫标记。该方法适用于各种不同来源和各种大小和形状的类器官。我们已经成功地将其用于人类气道，结肠，肾脏，肝脏和乳腺肿瘤类器官以及小鼠乳腺类器官。它包括一种简单的清除方法，利用自制的果糖-甘油清除剂，完全捕获3D类器官，并在细胞的基础上进行标记物定量。通过共聚焦、超分辨率共聚焦、多光子和光片显微镜，样品制备已被优化用于3D成像。从类器官收获到图像分析，整个过程需要3d。
In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.