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.
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We are very grateful for the technical support from the Princess Máxima Center for Pediatric Oncology and to the Hubrecht Institute and Zeiss for imaging support and collaborations. All the imaging was performed at the Princess Máxima imaging center. This work was financially supported by the Princess Máxima Center for Pediatric Oncology. J.F.D. was supported by a Marie Curie Global Fellowship and a VENI grant from the Netherlands Organisation for Scientific Research (NWO). J.E.V. was supported by the Australian National Health and Medical Research Council (NHMRC).
H.C. is named as inventor on several patents related to organoid technology. J.F.D. is named as inventor on one patent related to the organoid technology.
Journal peer review information: Nature Protocols thanks Xavier Gidrol, Melissa Skala and other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Hu, H. et al. Cell 175, 1591–1606.e19 (2018): https://doi.org/10.1016/j.cell.2018.11.013
Sachs, N. et al. EMBO J. 38, e100300 (2019): https://doi.org/10.15252/embj.2018100300
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Dekkers, J.F., Alieva, M., Wellens, L.M. et al. High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc 14, 1756–1771 (2019). https://doi.org/10.1038/s41596-019-0160-8
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