Journal Article

High-resolution 3D imaging of fixed and cleared organoids

Visvader, Jane E., Wehrens, Ellen J., Alieva, Maria, Vonk, Annelotte M., Clevers, Hans, Oost, Koen C., Ariese, Hendrikus C. R., Beekman, Jeffrey M., Snippert, Hugo J. G., Rios, Anne C., Wellens, Lianne M., Amatngalim, Gimano D., Dekkers, Johanna F., Hu, Huili, Jamieson, Paul R.

2019

Overview

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. This protocol for clearing and high-resolution 3D imaging of entire organoids expressing fluorescence reporters or following immunolabeling enables confocal, super-resolution confocal, multiphoton and light-sheet microscopy to be performed.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/245/2221 / 631/1647/245/2225 / 631/1647/245/2226 / 631/61/2320 / Analytical Chemistry / Animals / Biological Techniques / Biomedical and Life Sciences / Breast - ultrastructure / Breast cancer / Chemical microscopy / Chemical properties / Clearing / Colon / Colon - ultrastructure / Computational Biology/Bioinformatics / Female / Fluorescence / Fluorescence microscopy / Fructose / Glycerol / High resolution / Humans / Image analysis / Image processing / Image resolution / Imaging, Three-Dimensional - methods / Immunohistochemistry - methods / Kidney - ultrastructure / Kidneys / Life Sciences / Liver - ultrastructure / Mammary gland / Mammary glands / Methods / Mice / Microarrays / Microscopy / Microscopy, Confocal - methods / Microscopy, Fluorescence - methods / Optical Imaging - methods / Organic Chemistry / Organic compounds / Organoids / Organoids - ultrastructure / Protocol / Respiratory tract / Sample preparation / Three dimensional imaging / Three dimensional models / Tissue Fixation - methods