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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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.).
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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Aivia 3D: https://www.aivia-software.com/aivia-3d
- 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.
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 (2021). https://doi.org/10.1038/s41568-021-00382-w