Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht, Utrecht, the Netherlands.
Nat Protoc. 2019 Jun;14(6):1756-1771. doi: 10.1038/s41596-019-0160-8. Epub 2019 May 3.
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.
体外 3D 类器官系统在器官发育和疾病的体外建模方面带来了革命性的变化。荧光显微镜促进了类器官细胞组成的特征分析，并证明了类器官在表型上与其原始组织的相似性。本文提供了一个详细的方案，用于对含有荧光报告基因和免疫标记的整个 3D 类器官进行高分辨率成像。该方法适用于不同起源、不同大小和形状的各种类器官。我们已经成功地将其应用于人类气道、结肠、肾脏、肝脏和乳腺癌类器官，以及小鼠乳腺类器官。该方法包括一种简单的利用自制的果糖-甘油清除剂进行 3D 类器官全细胞成像的方法，该方法能够以单细胞为基础对标记物进行定量分析。该方案已经针对共聚焦、超分辨率共聚焦、多光子和光片显微镜的 3D 成像进行了优化。从类器官收获到图像分析，整个方案耗时 3 天。
