Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

You are viewing this page in draft mode.

Tissue clearing to examine tumour complexity in three dimensions

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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

References

  1. 1.

    Spalteholz, W. Über das Durchsichtigmachen von menschlichen und tierischen Präparaten und seine theoretischen Bedingungen [German] (S. Hirzel, 1914).

  2. 2.

    Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Voie, A. H., Burns, D. H. & Spelman, F. A. Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993).

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Ueda, H. R. et al. Tissue clearing and its applications in neuroscience. Nat. Rev. Neurosci. 21, 61–79 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Tedeschi, A. et al. Cep55 promotes cytokinesis of neural progenitors but is dispensable for most mammalian cell divisions. Nat. Commun. 11, 1746 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Dodt, H. U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Cabeza-Cabrerizo, M. et al. Tissue clonality of dendritic cell subsets and emergency DCpoiesis revealed by multicolor fate mapping of DC progenitors. Sci. Immunol. 4, eaaw1941 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Tanaka, N. et al. Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. Nat. Biomed. Eng. 1, 796–806 (2017). This study shows clearing of FFPE samples.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Kubota, S. I. et al. Whole-body profiling of cancer metastasis with single-cell resolution. Cell Rep. 20, 236–250 (2017). This study quantifies body-wide metastasis by whole mouse clearing.

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Garofalo, S. et al. Enriched environment reduces glioma growth through immune and non-immune mechanisms in mice. Nat. Commun. 6, 6623 (2015). This paper describes quantitative imaging of the lesion number and volume using tissue clearing.

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Pan, C. et al. Deep learning reveals cancer metastasis and therapeutic antibody targeting in the entire body. Cell 179, 1661–1676.e19 (2019). This paper describes deep learning-based segmentation of metastasis in whole mice.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Oshimori, N., Oristian, D. & Fuchs, E. TGF-β promotes heterogeneity and drug resistance in squamous cell carcinoma. Cell 160, 963–976 (2015). This paper identifies the EMT-inducing niche by tissue clearing.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Messal, H. A. et al. Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH. Nat. Protoc. 16, 239–262 (2021).

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Lagerweij, T. et al. Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment. Angiogenesis 20, 533–546 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Kingston, B. R., Syed, A. M., Ngai, J., Sindhwani, S. & Chan, W. C. W. Assessing micrometastases as a target for nanoparticles using 3D microscopy and machine learning. Proc. Natl Acad. Sci. USA 116, 14937–14946 (2019).

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Susaki, E. A. & Ueda, H. R. Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals. Cell Chem. Biol. 23, 137–157 (2016).

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Richardson, D. S. & Lichtman, J. W. Clarifying tissue clearing. Cell 162, 246–257 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T. & Ueda, H. R. Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu. Rev. Cell Dev. Biol. 32, 713–741 (2016).

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Ueda, H. R. et al. Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy. Neuron 106, 369–387 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Azaripour, A. et al. A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue. Prog. Histochem. Cytochem. 51, 9–23 (2016).

    PubMed  Article  Google Scholar 

  22. 22.

    Lloyd-Lewis, B. Multidimensional imaging of mammary gland development: a window into breast form and function. Front. Cell Dev. Biol. 8, 203 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Gomez-Gaviro, M. V., Sanderson, D., Ripoll, J. & Desco, M. Biomedical applications of tissue clearing and three-dimensional imaging in health and disease. iScience 23, 101432 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Susaki, E. A. et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat. Protoc. 10, 1709–1727 (2015).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Tainaka, K. et al. Whole-body imaging with single-cell resolution by tissue decolorization. Cell 159, 911–924 (2014).

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Sylwestrak, E. L., Rajasethupathy, P., Wright, M. A., Jaffe, A. & Deisseroth, K. Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell 164, 792–804 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Park, Y. G. et al. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat. Biotechnol. 37, 73–83 (2018).

    Article  CAS  Google Scholar 

  29. 29.

    Zhao, S. et al. Cellular and molecular probing of intact human organs. Cell 180, 796–812.e19 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Ke, M. T., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat. Neurosci. 16, 1154–1161 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  31. 31.

    Ke, M. T. et al. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent. Cell Rep. 14, 2718–2732 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    Kuwajima, T. et al. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140, 1364–1368 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Hama, H. et al. ScaleS: an optical clearing palette for biological imaging. Nat. Neurosci. 18, 1518–1529 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Pan, C. et al. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13, 859–867 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Jing, D. et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 28, 803–818 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Schwarz, M. K. et al. Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains. PLoS ONE 10, e0124650 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  37. 37.

    Qi, Y. et al. FDISCO: advanced solvent-based clearing method for imaging whole organs. Sci. Adv. 5, eaau8355 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. 38.

    Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Hofmann, J., Gadjalova, I., Mishra, R., Ruland, J. & Keppler, S. J. Efficient tissue clearing and multi-organ volumetric imaging enable quantitative visualization of sparse immune cell populations during inflammation. Front. Immunol. 11, 599495 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Erturk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat. Protoc. 7, 1983–1995 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41.

    Erturk, A. et al. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat. Med. 18, 166–171 (2012).

    Article  CAS  Google Scholar 

  42. 42.

    Tainaka, K. et al. Chemical landscape for tissue clearing based on hydrophilic reagents. Cell Rep. 24, 2196–2210.e9 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Messal, H. A. et al. Tissue curvature and apicobasal mechanical tension imbalance instruct cancer morphogenesis. Nature 566, 126–130 (2019). This paper identifies epithelial geometry-driven tumour initiation and progression by tissue clearing.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Chen, L. et al. UbasM: an effective balanced optical clearing method for intact biomedical imaging. Sci. Rep. 7, 12218 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. 46.

    Chi, J. et al. Three-dimensional adipose tissue imaging reveals regional variation in beige fat biogenesis and PRDM16-dependent sympathetic neurite density. Cell Metab. 27, 226–236.e3 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Li, W., Germain, R. N. & Gerner, M. Y. Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D). Proc. Natl Acad. Sci. USA 114, E7321–E7330 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Treweek, J. B. et al. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat. Protoc. 10, 1860–1896 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Minsky, M. Microscopy apparatus. US Patent 3,013,467 (1961).

  50. 50.

    Sharpe, J. et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    d’Esposito, A. et al. Computational fluid dynamics with imaging of cleared tissue and of in vivo perfusion predicts drug uptake and treatment responses in tumours. Nat. Biomed. Eng. 2, 773–787 (2018).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  52. 52.

    Lloyd-Lewis, B. et al. Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods. Breast Cancer Res. 18, 127 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Li, S., Gestl, S. A. & Gunther, E. J. A multistage murine breast cancer model reveals long-lived premalignant clones refractory to parity-induced protection. Cancer Prev. Res. 13, 173–184 (2020).

    CAS  Article  Google Scholar 

  54. 54.

    Wei, M. et al. Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy. Proc. Natl Acad. Sci. USA 116, 6608–6617 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Reynaud, E. G., Krzic, U., Greger, K. & Stelzer, E. H. Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage. HFSP J. 2, 266–275 (2008).

    PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Herbert, S. P. et al. Arterial-venous segregation by selective cell sprouting: an alternative mode of blood vessel formation. Science 326, 294–298 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Power, R. M. & Huisken, J. A guide to light-sheet fluorescence microscopy for multiscale imaging. Nat. Methods 14, 360–373 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  58. 58.

    Sabdyusheva Litschauer, I. et al. 3D histopathology of human tumours by fast clearing and ultramicroscopy. Sci. Rep. 10, 17619 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 0084 (2017). This study develops 3D haematoxylin and eosin-like staining.

    PubMed  PubMed Central  Article  Google Scholar 

  60. 60.

    Tian, T., Yang, Z. & Li, X. Tissue clearing technique: recent progress and biomedical applications. J. Anat. 238, 489–507 (2021).

    PubMed  Article  PubMed Central  Google Scholar 

  61. 61.

    Tomer, R., Ye, L., Hsueh, B. & Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat. Protoc. 9, 1682–1697 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. 62.

    Belle, M. et al. Tridimensional visualization and analysis of early human development. Cell 169, 161–173.e12 (2017).

    CAS  PubMed  Article  Google Scholar 

  63. 63.

    Matsumoto, K. et al. Advanced CUBIC tissue clearing for whole-organ cell profiling. Nat. Protoc. 14, 3506–3537 (2019).

    CAS  PubMed  Article  Google Scholar 

  64. 64.

    Murakami, T. C. et al. A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat. Neurosci. 21, 625–637 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  65. 65.

    Chakraborty, T. et al. Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution. Nat. Methods 16, 1109–1113 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. 66.

    Petran, M., Hadravský, M., Egger, M. D. & Galambos, R. Tandem-scanning reflected-light microscope. J. Optical Soc. Am. 58, 661–664 (1968).

    Article  Google Scholar 

  67. 67.

    Jonkman, J. & Brown, C. M. Any way you slice it — a comparison of confocal microscopy techniques. J. Biomol. Tech. 26, 54–65 (2015).

    PubMed  PubMed Central  Article  Google Scholar 

  68. 68.

    Wu, Y. et al. Resonant scanning with large field of view reduces photobleaching and enhances fluorescence yield in STED microscopy. Sci. Rep. 5, 14766 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. 69.

    Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  70. 70.

    Lin, P. Y., Peng, S. J., Shen, C. N., Pasricha, P. J. & Tang, S. C. PanIN-associated pericyte, glial, and islet remodeling in mice revealed by 3D pancreatic duct lesion histology. Am. J. Physiol. Gastrointest. Liver Physiol. 311, G412–G422 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  71. 71.

    Lay, K. et al. Stem cells repurpose proliferation to contain a breach in their niche barrier. eLife 7, e41661 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  72. 72.

    Fiore, V. F. et al. Mechanics of a multilayer epithelium instruct tumour architecture and function. Nature 585, 433–439 (2020). This paper identifies early tumour architecture in complex epithelia by tissue clearing.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. 73.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Article  Google Scholar 

  74. 74.

    Liebmann, T. et al. Three-dimensional study of Alzheimer’s disease hallmarks using the iDISCO clearing method. Cell Rep. 16, 1138–1152 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  75. 75.

    Casoni, F. et al. Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains. Development 143, 3969–3981 (2016).

    CAS  PubMed  Article  Google Scholar 

  76. 76.

    Belle, M. et al. A simple method for 3D analysis of immunolabeled axonal tracts in a transparent nervous system. Cell Rep. 9, 1191–1201 (2014).

    CAS  PubMed  Article  Google Scholar 

  77. 77.

    Breckwoldt, M. O. et al. Correlated magnetic resonance imaging and ultramicroscopy (MR-UM) is a tool kit to assess the dynamics of glioma angiogenesis. eLife 5, e11712 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  78. 78.

    McIlwain, H. & Bachelard, H. S. Biochemistry and the Central Nervous System (Churchill Livingstone, 1985).

  79. 79.

    Oldham, M., Sakhalkar, H., Oliver, T., Allan Johnson, G. & Dewhirst, M. Optical clearing of unsectioned specimens for three-dimensional imaging via optical transmission and emission tomography. J. Biomed. Opt. 13, 021113 (2008).

    PubMed  Article  Google Scholar 

  80. 80.

    Sung, K. et al. Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping. Sci. Rep. 6, 30736 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  81. 81.

    Cancer Research UK. Types of cancer. Cancer Research UK https://www.cancerresearchuk.org/what-is-cancer/how-cancer-starts/types-of-cancer (2020).

  82. 82.

    Fu, Y. Y. et al. Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution. Gastroenterology 137, 453–465 (2009).

    PubMed  Article  Google Scholar 

  83. 83.

    Liu, Y. A. et al. 3-D imaging, illustration, and quantitation of enteric glial network in transparent human colon mucosa. Neurogastroenterol. Motil. 25, e324–e338 (2013).

    CAS  PubMed  Article  Google Scholar 

  84. 84.

    Bernier-Latmani, J. & Petrova, T. V. High-resolution 3D analysis of mouse small-intestinal stroma. Nat. Protoc. 11, 1617–1629 (2016).

    CAS  PubMed  Article  Google Scholar 

  85. 85.

    Liu, Y. A. et al. Perivascular interstitial cells of cajal in human colon. Cell Mol. Gastroenterol. Hepatol. 1, 102–119 (2015).

    PubMed  Article  Google Scholar 

  86. 86.

    Davis, F. M. et al. Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny. Nat. Commun. 7, 13053 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  87. 87.

    Rios, A. C. et al. Intraclonal plasticity in mammary tumors revealed through large-scale single-cell resolution 3D imaging. Cancer Cell 35, 618–632.e6 (2019). This paper identifies the EMT-inducing niche by tissue clearing.

    CAS  Article  Google Scholar 

  88. 88.

    Cai, R. et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nat. Neurosci. 22, 317–327 (2019).

    CAS  PubMed  Article  Google Scholar 

  89. 89.

    Gur-Cohen, S. et al. Stem cell-driven lymphatic remodeling coordinates tissue regeneration. Science 366, 1218–1225 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  90. 90.

    Tang, S. C. et al. Pancreatic neuro-insular network in young mice revealed by 3D panoramic histology. Diabetologia 61, 158–167 (2018).

    PubMed  Article  Google Scholar 

  91. 91.

    Hong, S. M. et al. Three-dimensional visualization of cleared human pancreas cancer reveals that sustained epithelial-to-mesenchymal transition is not required for venous invasion. Mod. Pathol. 33, 639–647 (2020).

    CAS  PubMed  Article  Google Scholar 

  92. 92.

    Gradauer, K. et al. Interaction with mixed micelles in the intestine attenuates the permeation enhancing potential of alkyl-maltosides. Mol. Pharm. 12, 2245–2253 (2015).

    CAS  PubMed  Article  Google Scholar 

  93. 93.

    Hu, H. et al. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell 175, 1591–1606.e19 (2018).

    CAS  PubMed  Article  Google Scholar 

  94. 94.

    Sachs, N. et al. Long-term expanding human airway organoids for disease modeling. EMBO J. 38, e100300 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  95. 95.

    Dekkers, J. F. et al. High-resolution 3D imaging of fixed and cleared organoids. Nat. Protoc. 14, 1756–1771 (2019).

    CAS  PubMed  Article  Google Scholar 

  96. 96.

    Grist, S. M., Nasseri, S. S., Poon, T., Roskelley, C. & Cheung, K. C. On-chip clearing of arrays of 3-D cell cultures and micro-tissues. Biomicrofluidics 10, 044107 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  97. 97.

    van Royen, M. E. et al. Three-dimensional microscopic analysis of clinical prostate specimens. Histopathology 69, 985–992 (2016).

    PubMed  Article  Google Scholar 

  98. 98.

    Noe, M. et al. Immunolabeling of cleared human pancreata provides insights into three-dimensional pancreatic anatomy and pathology. Am. J. Pathol. 188, 1530–1535 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  99. 99.

    Guldner, I. H. et al. An integrative platform for three-dimensional quantitative analysis of spatially heterogeneous metastasis landscapes. Sci. Rep. 6, 24201 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  100. 100.

    Brown, M. et al. Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice. Science 359, 1408–1411 (2018).

    CAS  PubMed  Article  Google Scholar 

  101. 101.

    Pereira, E. R. et al. Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice. Science 359, 1403–1407 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  102. 102.

    Song, E. et al. Optical clearing based cellular-level 3D visualization of intact lymph node cortex. Biomed. Opt. Express 6, 4154–4164 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  103. 103.

    Nojima, S. et al. CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci. Rep. 7, 9269 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  104. 104.

    von Neubeck, B. et al. An inhibitory antibody targeting carbonic anhydrase XII abrogates chemoresistance and significantly reduces lung metastases in an orthotopic breast cancer model in vivo. Int. J. Cancer 143, 2065–2075 (2018).

    Article  CAS  Google Scholar 

  105. 105.

    Yang, R. et al. The combination of two-dimensional and three-dimensional analysis methods contributes to the understanding of glioblastoma spatial heterogeneity. J. Biophotonics 13, e201900196 (2020).

    PubMed  Google Scholar 

  106. 106.

    Liu, Y. A. et al. 3-D visualization and quantitation of microvessels in transparent human colorectal carcinoma [corrected]. PLoS ONE 8, e81857 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  107. 107.

    Tanaka, N. et al. Mapping of the three-dimensional lymphatic microvasculature in bladder tumours using light-sheet microscopy. Br. J. Cancer 118, 995–999 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  108. 108.

    Mendler, C. T. et al. Tumor uptake of anti-CD20 Fabs depends on tumor perfusion. J. Nucl. Med. 57, 1971–1977 (2016).

    CAS  PubMed  Article  Google Scholar 

  109. 109.

    Dobosz, M., Ntziachristos, V., Scheuer, W. & Strobel, S. Multispectral fluorescence ultramicroscopy: three-dimensional visualization and automatic quantification of tumor morphology, drug penetration, and antiangiogenic treatment response. Neoplasia 16, 1–13 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  110. 110.

    Poschinger, T. et al. Dynamic contrast-enhanced micro-computed tomography correlates with 3-dimensional fluorescence ultramicroscopy in antiangiogenic therapy of breast cancer xenografts. Invest. Radiol. 49, 445–456 (2014).

    PubMed  Article  CAS  Google Scholar 

  111. 111.

    Si, Y. et al. Multidimensional imaging provides evidence for down-regulation of T cell effector function by MDSC in human cancer tissue. Sci. Immunol. 4, aaw9159 (2019). This paper identifies neutrophil hotspots that induce immune evasion by tissue clearing.

    Article  CAS  Google Scholar 

  112. 112.

    Chen, Y. et al. Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues. Sci. Rep. 9, 5624 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  113. 113.

    Lee, S. S., Bindokas, V. P. & Kron, S. J. Multiplex three-dimensional optical mapping of tumor immune microenvironment. Sci. Rep. 7, 17031 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  114. 114.

    Brown, A. S. et al. Histologic changes associated with false-negative sentinel lymph nodes after preoperative chemotherapy in patients with confirmed lymph node-positive breast cancer before treatment. Cancer 116, 2878–2883 (2010).

    PubMed  Article  Google Scholar 

  115. 115.

    Yokota, T. et al. Accuracy of preoperative diagnosis of lymph node metastasis for thoracic esophageal cancer patients from JCOG9907 trial. Int. J. Clin. Oncol. 21, 283–288 (2016).

    CAS  PubMed  Article  Google Scholar 

  116. 116.

    Frechet, B., Kazakov, J., Thiffault, V., Ferraro, P. & Liberman, M. Diagnostic accuracy of mediastinal lymph node staging techniques in the preoperative assessment of nonsmall cell lung cancer patients. J. Bronchol. Interv. Pulmonol. 25, 17–24 (2018).

    Article  Google Scholar 

  117. 117.

    King, C. R. & Long, J. P. Prostate biopsy grading errors: a sampling problem? Int. J. Cancer 90, 326–330 (2000).

    CAS  PubMed  Article  Google Scholar 

  118. 118.

    Catalona, W. J., Stein, A. J. & Fair, W. R. Grading errors in prostatic needle biopsies: relation to the accuracy of tumor grade in predicting pelvic lymph node metastases. J. Urol. 127, 919–922 (1982).

    CAS  PubMed  Article  Google Scholar 

  119. 119.

    Epstein, J. I. Prostate cancer grading: a decade after the 2005 modified system. Mod. Pathol. 31, S47–S63 (2018).

    Article  Google Scholar 

  120. 120.

    Ahdoot, M. et al. MRI-targeted, systematic, and combined biopsy for prostate cancer diagnosis. N. Engl. J. Med. 382, 917–928 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  121. 121.

    Bostrom, P. J. et al. Staging and staging errors in bladder cancer. Eur. Urol. Suppl. 9, 2–9 (2010).

    Article  Google Scholar 

  122. 122.

    Kruskal, J. B., Kane, R. A., Sentovich, S. M. & Longmaid, H. E. Pitfalls and sources of error in staging rectal cancer with endorectal us. Radiographics 17, 609–626 (1997).

    CAS  PubMed  Article  Google Scholar 

  123. 123.

    Torres, R., Vesuna, S. & Levene, M. J. High-resolution, 2- and 3-dimensional imaging of uncut, unembedded tissue biopsy samples. Arch. Pathol. Lab. Med. 138, 395–402 (2014).

    PubMed  Article  Google Scholar 

  124. 124.

    Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  125. 125.

    Hsueh, B. et al. Pathways to clinical CLARITY: volumetric analysis of irregular, soft, and heterogeneous tissues in development and disease. Sci. Rep. 7, 5899 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  126. 126.

    Tanaka, N. et al. Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies. Nat. Biomed. Eng. 4, 875–888 (2020).

    CAS  PubMed  Article  Google Scholar 

  127. 127.

    Sun, D. E. et al. Click-ExM enables expansion microscopy for all biomolecules. Nat. Methods 18, 107–113 (2021).

    CAS  PubMed  Article  Google Scholar 

  128. 128.

    Liu, J. T. C. et al. Harnessing non-destructive 3D pathology. Nat. Biomed. Eng. 5, 203–218 (2021).

    PubMed  Article  Google Scholar 

  129. 129.

    Eisenstein, M. Transparent tissues bring cells into focus for microscopy. Nature 564, 147–149 (2018).

    CAS  PubMed  Article  Google Scholar 

  130. 130.

    Franca, C. M. et al. 3D-imaging of whole neuronal and vascular networks of the human dental pulp via CLARITY and light sheet microscopy. Sci. Rep. 9, 10860 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  131. 131.

    Hook, P. et al. Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis. Biomed. Opt. Express 8, 3671–3686 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  132. 132.

    Bulantova, J. et al. Trichobilharzia regenti (Schistosomatidae): 3D imaging techniques in characterization of larval migration through the CNS of vertebrates. Micron 83, 62–71 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  133. 133.

    Kang, G. Y., Rhyu, H. J., Choi, H. H., Shin, S. J. & Hyun, Y. M. 3D Imaging of the transparent Mycobacterium tuberculosis-infected lung verifies the localization of innate immune cells with granuloma. Front. Cell Infect. Microbiol. 10, 226 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  134. 134.

    Zaeck, L. M. et al. 3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract. Preprint at bioRxiv https://doi.org/10.1101/2020.10.17.339051 (2020).

    Article  Google Scholar 

  135. 135.

    Wang, H. et al. Deep learning enables cross-modality super-resolution in fluorescence microscopy. Nat. Methods 16, 103–110 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  136. 136.

    Murray, E. et al. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163, 1500–1514 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  137. 137.

    Hou, B. et al. Scalable and DiI-compatible optical clearance of the mammalian brain. Front. Neuroanat. 9, 19 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  138. 138.

    Cai, R. et al. Panoptic vDISCO imaging reveals neuronal connectivity, remote trauma effects and meningeal vessels in intact transparent mice. Preprint at bioRxiv https://doi.org/10.1101/374785 (2018).

    Article  Google Scholar 

  139. 139.

    Masselink, W. et al. Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development 146, dev166884 (2019).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  140. 140.

    Hahn, C. et al. High-resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO). J. Biophotonics 12, e201800368 (2019).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  141. 141.

    Tseng, S. J. et al. Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections. J. Biomed. Opt. 14, 044004 (2009).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  142. 142.

    Liu, K. et al. Metabolic stress drives sympathetic neuropathy within the liver. Cell Metab. 33, 666–675.e4 (2021).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  143. 143.

    Chen, F., Tillberg, P. & Boyden, E. Expansion microscopy. Science 347, 543–548 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  144. 144.

    Tillberg, P. W. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 34, 987–992 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  145. 145.

    Miyabayashi, K. et al. Intraductal transplantation models of human pancreatic ductal adenocarcinoma reveal progressive transition of molecular subtypes. Cancer Discov. 10, 1566–1589 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  146. 146.

    Li, W., Germain, R. N. & Gerner, M. Y. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat. Protoc. 14, 1708–1733 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  147. 147.

    Greenbaum, A. et al. Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci. Transl. Med. 9, eaah6518 (2017).

    PubMed  Article  Google Scholar 

  148. 148.

    van Ineveld, R. et al. Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-00926-3 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  149. 149.

    Kugler, R. et al. Novel imaging of the prostate reveals spontaneous gland contraction and excretory duct quiescence together with different drug effects. FASEB J. 32, 1130–1138 (2018).

    PubMed  Article  PubMed Central  Google Scholar 

  150. 150.

    LeCun, Y., Bengio, Y. & Hinton, G. Deep learning. Nature 521, 436–444 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  151. 151.

    Todorov, M. I. et al. Machine learning analysis of whole mouse brain vasculature. Nat. Methods 17, 442–449 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  152. 152.

    Berg, S. et al. ilastik: interactive machine learning for (bio)image analysis. Nat. Methods 16, 1226–1232 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  153. 153.

    Schoppe, O. et al. Deep learning-enabled multi-organ segmentation in whole-body mouse scans. Nat. Commun. 11, 5626 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  154. 154.

    Isensee, F., Jaeger, P. F., Kohl, S. A. A., Petersen, J. & Maier-Hein, K. H. nnU-Net: a self-configuring method for deep learning-based biomedical image segmentation. Nat. Methods 18, 203–211 (2021).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  155. 155.

    Sullivan, D. P. et al. Deep learning is combined with massive-scale citizen science to improve large-scale image classification. Nat. Biotechnol. 36, 820–828 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  156. 156.

    McKinney, S. M. et al. International evaluation of an AI system for breast cancer screening. Nature 577, 89–94 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  157. 157.

    Savage, N. How AI is improving cancer diagnostics. Nature 579, S14–S16 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  158. 158.

    Paluch, E. K. After the greeting: realizing the potential of physical models in cell biology. Trends Cell Biol. 25, 711–713 (2015).

    PubMed  Article  PubMed Central  Google Scholar 

Download references

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.).

Author information

Affiliations

Authors

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.

Ethics declarations

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.

Additional information

Peer review information

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.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing