High-resolution 3D imaging of fixed and cleared organoids

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

doi:10.1038/s41596-019-0160-8

High-resolution 3D imaging of fixed and cleared organoids

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

Hubrecht Institute

Research output: Contribution to journal/periodical › Article › Scientific › peer-review

305 Citations (Scopus)

Abstract

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.

Original language	English
Pages (from-to)	1756-1771
Number of pages	16
Journal	Nature Protocols
Volume	14
Issue number	6
DOIs	https://doi.org/10.1038/s41596-019-0160-8
Publication status	Published - Jun 2019

Keywords

Animals
Breast/ultrastructure
Colon/ultrastructure
Female
Humans
Imaging, Three-Dimensional/methods
Immunohistochemistry/methods
Kidney/ultrastructure
Liver/ultrastructure
Mice
Microscopy, Confocal/methods
Microscopy, Fluorescence/methods
Optical Imaging/methods
Organoids/ultrastructure
Tissue Fixation/methods

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10.1038/s41596-019-0160-8

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Dekkers, J. F., Alieva, M., Wellens, L. M., Ariese, H. C. R., Jamieson, P. R., Vonk, A. M., Amatngalim, G. D., Hu, H., Oost, K. C., Snippert, H. J. G., Beekman, J. M., Wehrens, E. J., Visvader, J. E., Clevers, H., & Rios, A. C. (2019). High-resolution 3D imaging of fixed and cleared organoids. Nature Protocols, 14(6), 1756-1771. https://doi.org/10.1038/s41596-019-0160-8

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abstract = "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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AU - Vonk, Annelotte M

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AU - Snippert, Hugo J G

AU - Beekman, Jeffrey M

AU - Wehrens, Ellen J

AU - Visvader, Jane E

AU - Clevers, Hans

AU - Rios, Anne C

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KW - Liver/ultrastructure

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KW - Microscopy, Confocal/methods

KW - Microscopy, Fluorescence/methods

KW - Optical Imaging/methods

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JO - Nature Protocols

JF - Nature Protocols

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High-resolution 3D imaging of fixed and cleared organoids

Abstract

Keywords

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