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High-resolution 3D imaging of fixed and cleared organoids


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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Fig. 1: Overview of the high-resolution 3D imaging protocol.
Fig. 2: Representative 3D whole-mount organoid images obtained with different light microscopy technologies.
Fig. 3: Comparison of different optical clearing agents and single- and multiphoton imaging.
Fig. 4: 3D imaging enables acquisition of accurate architectural information and 3D quantification.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).


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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).

Author information




J.F.D. designed the study, performed experiments and interpreted data; J.F.D., M.A., L.M.W., H.C.R.A. and P.R.J. performed experiments and analyzed data; J.F.D., P.R.J., A.M.V., G.D.A., H.H. and J.M.B. performed the organoid culturing. K.C.O. and H.J.G.S. provided fluorescent constructs; A.C.R. helped design the study and carried out data interpretation; J.F.D. and A.C.R. cowrote the manuscript. J.E.V., H.C. and E.J.W. helped with data interpretation, manuscript writing and corrections.

Corresponding author

Correspondence to Anne C. Rios.

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Competing interests

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.

Additional information

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.

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Key references using this protocol

Hu, H. et al. Cell 175, 1591–1606.e19 (2018):

Sachs, N. et al. EMBO J. 38, e100300 (2019):

Supplementary information

Supplementary Video 1

This video highlights the intricate 3D features of delicate organoid structures that can be imaged at cellular or even subcellular resolution with this sample-preparation protocol.

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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).

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