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  • Review Article
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Tissue clearing to examine tumour complexity in three dimensions

Nature Reviews Cancer volume 21, pages 718–730 (2021)Cite this article

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Abstract

The visualization of whole organs and organisms through tissue clearing and fluorescence volumetric imaging has revolutionized the way we look at biological samples. Its application to solid tumours is changing our perception of tumour architecture, revealing signalling networks and cell interactions critical in tumour progression, and provides a powerful new strategy for cancer diagnostics. This Review introduces the latest advances in tissue clearing and three-dimensional imaging, examines the challenges in clearing epithelia — the tissue of origin of most malignancies — and discusses the insights that tissue clearing has brought to cancer research, as well as the prospective applications to experimental and clinical oncology.

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Fig. 1: General steps for tissue permeabilization, clearing and 3D imaging.
Fig. 2: Imaging approaches for transparent tissues.

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Acknowledgements

The authors are grateful to K. Ng (The Francis Crick Institute), R. Ng (University of British Columbia), A. Neumann (The Francis Crick Institute and Imperial College London) and C. Basier (The Francis Crick Institute) for critical reading of the manuscript. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001039, FC001317), the UK Medical Research Council (FC001039, FC001317), the Wellcome Trust (FC001039, FC001317), the European Molecular Biology Organization (EMBO long-term fellowship ALTF 452-2019 to H.A.M.) and the Doctor Josef Steiner Foundation (to J.v.R.).

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Author notes

  1. These authors contributed equally: Jorge Almagro, Hendrik A. Messal.

Authors and Affiliations

  1. Adult Stem Cell Laboratory, The Francis Crick Institute, London, UK

    Jorge Almagro & Axel Behrens

  2. Department of Molecular Pathology, Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands

    Hendrik A. Messal & Jacco van Rheenen

  3. Cancer Stem Cell Laboratory, Institute of Cancer Research, London, UK

    May Zaw Thin & Axel Behrens

  4. Convergence Science Centre and Division of Cancer, Department of Surgery and Cancer, Imperial College London, London, UK

    Axel Behrens

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  1. Jorge Almagro
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  2. Hendrik A. Messal
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  4. Jacco van Rheenen
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Contributions

J.A., H.A.M. and M.Z.T. researched data for the article, made substantial contribution to discussion of content and wrote, reviewed and edited the manuscript before submission. A.B. and J.v.R. made substantial contribution to discussion of content and wrote, reviewed and edited the manuscript before submission.

Corresponding author

Correspondence to Axel Behrens.

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Competing interests

H.A.M. and A.B. are inventors on a UK patent application (1818567.8) on clearing solutions for tissue clearing and three-dimensional (3D) imaging. The other authors declare no competing interests.

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Nature Reviews Cancer thanks Ali Erturk, who co-reviewed with Chenchen Pan, Hiroki Ueda and Per Uhlén for their contribution to the peer review of this work.

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Related links

Aivia 3D: https://www.aivia-software.com/aivia-3d

Imaris: https://imaris.oxinst.com

Vision4D: https://imaging.arivis.com/en/imaging-science/arivis-vision4d

Supplementary information

Glossary

Light-sheet fluorescence microscopy

(LSFM). A fluorescence microscopy technique in which only a plane of the sample is illuminated at one time.

Numerical aperture

A dimensionless number proportional to the refractive index of the medium and the sine of the maximum half-angle of light passing through a lens.

Raman scattering microscopy

A technique to image chemical bonds in biological samples by quantum amplification via stimulated emission.

Point scanning confocal microscopes

Fluorescence microscopes that illuminate a single point of the sample and use a pinhole to eliminate the out of focus signal.

Axially swept light-sheet microscopy

A light-sheet fluorescence microscopy technique in which illumination is scanned in its direction of propagation.

Free working distance

The distance from the front of the objective to the closest surface of the sample in focus.

Spinning disk confocal microscopy

A confocal microscopy technique that uses multiple pinholes on a spinning disk to direct the excitation and emission light beams and increase the imaging speed.

Resonant scanning confocal microscopy

A confocal microscopy technique with galvanometric mirrors for fast image acquisition.

Vibratome

An instrument that slices micrometre-scale sample sections with a vibrating blade.

Expansion microscopy

(ExM). A sample preparation protocol in which specimens are isometrically swollen in a polymer gel to image small structures.

Click chemistry

A biocompatible reaction allowing covalent bonding of a biomolecule to a substrate of choice.

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Almagro, J., Messal, H.A., Zaw Thin, M. et al. Tissue clearing to examine tumour complexity in three dimensions. Nat Rev Cancer 21, 718–730 (2021). https://doi.org/10.1038/s41568-021-00382-w

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