Medicine:Tissue clearing

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Tissue clearing refers to a group of chemical techniques used to turn tissues transparent.[1][2][3] This allows deep insight into these tissues, while preserving spatial resolution.[1] Many tissue clearing methods exist, each with different strengths and weaknesses.[2][4] Some are generally applicable, while others are designed for specific applications.[4] Tissue clearing is usually combined with one or more labeling techniques and subsequently imaged, most often by optical sectioning microscopy techniques.[1][5][6] Tissue clearing has been applied to many areas in biological research.[7]

History

In the early 1900s, Werner Spalteholz developed a technique that allowed the clarification of large tissues,[2][8] using Wintergrünöl (methyl salicylate) and benzyl benzoate.[9] Over the next hundred years, various scientists introduced their own variations on Spalteholz's technique.[8] Tuchin et al. introduced TOC in 1997, adding a new branch of tissue clearing that was hydrophilic instead of hydrophobic like Spalteholz's technique.[1][10] In 2007, Dodt et al. developed a two step process, wherein tissues were first dehydrated with ethanol and hexane and subsequently made transparent by immersion in benzyl alcohol and benzyl benzoate (BABB), a technique they coupled with light sheet fluorescence microscopy.[2][3] Hama et al. developed another hydrophilic approach, Scale, in 2011.[2][8] The following year, Ertürk et al. developed a hydrophobic approach called 3DISCO, in which they pretreated tissue with tetrahydrofuran and dichloromethane before clearing it in dibenzyl ether.[3][8] A year later, in 2013, Chung et al. developed CLARITY, the first approach to use hydrogel monomers to clear tissue.[2][3][8]

Principles

Tissue opacity is thought to be the result of light scattering due to heterogeneous refractive indices.[1][4][5] Tissue clearing methods chemically homogenize refractive indices, resulting in almost completely transparent tissue.[4][6]

Classifications

While multiple classification standards for tissue clearing exist, the most common classifications use the chemical principle and mechanism of clearing to group tissue clearing methods.[1] These include hydrophobic clearing methods,[1][2][6] which may also be known as organic,[3] solvent-based,[4][5] organic solvent-based,[11][12] or dehydration[13] clearing methods; hydrophilic clearing methods,[1][2][6] which may also be known as aqueous-based[5][11] or water-based[13] methods, and may be further sub-categorized into simple immersion[4] and hyperhydration[4] (also called delipidation/hydation[5]); and hydrogel-based clearing methods, which may also be known as detergent[3] or hydrogel embedding[4][5][11] methods. Tissue-expansion clearing methods use hydrogel, and may be included under hydrogel-based clearing[2] or as their own category.[1]

Methods

Common methods include those of the DISCO family, including 3DISCO, and CLARITY and related protocols.[1][2][3] Others include BABB,[1][2][4] PEGASOS,[1][2][4] SHANEL,[1][5] SeeDB,[1][2][4] CUBIC,[1][2][4] ExM,[1][2][4] and SHIELD.[1][4][5]

Labeling

Tissue clearing methods have varying compatibility with different methods of fluorescent labeling.[1][5][6] Some are better suited to pre-clearing tagging approaches, such as genetic labeling.[1][5] while others require post-clearing tagging, such as immunolabeling and chemical dye labeling.[1][5]

Imaging

After clearing and labeling, tissues are typically imaged using confocal microscopy,[11][12][13] two-photon microscopy,[1][5][11] or one of the many variants of light-sheet fluorescence microscopy.[7][11][12] Other less commonly used methods include optical projection tomography[1][5] and stimulated Raman scattering.[5][7][11]

Data

Imaging cleared tissues generates massive volumes of complex data, which requires powerful computational hardware and software to store, process, analyze, and visualize.[1][6][13] A single mouse brain can generate terabytes of data.[2][6][13] Both commercial and open-source software exists to address this need, some of it adapted from solutions for two-dimensional images and some of it designed specifically for the three-dimensional images produced by imaging of cleared tissues.[1][11][12]

Applications

Tissue clearing has been applied to the nervous system,[1][2][3][4][5][6][7][11][14][15] bones (including teeth),[7][11][12][16][17][18] skeletal muscles,[7][18][19] hearts and vasculature,[7][11][20] gastrointestinal organs,[7][21] urogenital organs,[7][11][22] skin,[7][23] lymph nodes,[7] mammary glands,[7] lungs,[7] eyes,[7] tumors,[7][11] and adipose tissues.[7][11] Whole-body clearing is less common, but has been done in smaller animals, including rodents.[1][6][7] Tissue clearing has also been applied to human cancer tissues [24][25]

References

  1. ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 1.19 1.20 1.21 1.22 1.23 1.24 1.25 "Current Status of Tissue Clearing and the Path Forward in Neuroscience". ACS Chemical Neuroscience 12 (1): 5–29. January 2021. doi:10.1021/acschemneuro.0c00563. PMID 33326739. 
  2. ↑ 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 2.15 2.16 "Whole-Brain Profiling of Cells and Circuits in Mammals by Tissue Clearing and Light-Sheet Microscopy". Neuron 106 (3): 369–387. May 2020. doi:10.1016/j.neuron.2020.03.004. PMID 32380050. 
  3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 "Neuroscience in the third dimension: shedding new light on the brain with tissue clearing". Molecular Brain 10 (1): 33. July 2017. doi:10.1186/s13041-017-0314-y. PMID 28728585. 
  4. ↑ 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 "Clearing techniques for visualizing the nervous system in development, injury, and disease". Journal of Neuroscience Methods 334: 108594. January 2020. doi:10.1016/j.jneumeth.2020.108594. PMID 31945400. 
  5. ↑ 5.00 5.01 5.02 5.03 5.04 5.05 5.06 5.07 5.08 5.09 5.10 5.11 5.12 5.13 5.14 "Applications of tissue clearing in the spinal cord". The European Journal of Neuroscience 52 (9): 4019–4036. November 2020. doi:10.1111/ejn.14938. PMID 32794596. 
  6. ↑ 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 "Tissue clearing and its applications in neuroscience". Nature Reviews. Neuroscience 21 (2): 61–79. February 2020. doi:10.1038/s41583-019-0250-1. PMID 31896771. 
  7. ↑ 7.00 7.01 7.02 7.03 7.04 7.05 7.06 7.07 7.08 7.09 7.10 7.11 7.12 7.13 7.14 7.15 7.16 "Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease" (in English). iScience 23 (8): 101432. August 2020. doi:10.1016/j.isci.2020.101432. PMID 32805648. Bibcode: 2020iSci...23j1432G. 
  8. ↑ 8.0 8.1 8.2 8.3 8.4 "A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue". Progress in Histochemistry and Cytochemistry 51 (2): 9–23. August 2016. doi:10.1016/j.proghi.2016.04.001. PMID 27142295. 
  9. ↑ Über das Durchsichtigmachen von menschlichen und tierischen Präparaten und seine theoretischen Bedingungen, nebst Anhang: Über Knochenfärbung. Leipzig: S. Hirzel. 1914. 
  10. ↑ "Light propagation in tissues with controlled optical properties". Journal of Biomedical Optics 2 (4): 401–17. October 1997. doi:10.1117/12.281502. PMID 23014964. Bibcode: 1997JBO.....2..401T. 
  11. ↑ 11.00 11.01 11.02 11.03 11.04 11.05 11.06 11.07 11.08 11.09 11.10 11.11 11.12 11.13 "Tissue clearing technique: Recent progress and biomedical applications". Journal of Anatomy 238 (2): 489–507. February 2021. doi:10.1111/joa.13309. PMID 32939792. 
  12. ↑ 12.0 12.1 12.2 12.3 12.4 "Tissue Clearing and Its Application to Bone and Dental Tissues". Journal of Dental Research 98 (6): 621–631. June 2019. doi:10.1177/0022034519844510. PMID 31009584. 
  13. ↑ 13.0 13.1 13.2 13.3 13.4 "Massive volumetric imaging of cleared tissue: The necessary tools to be successful". The International Journal of Biochemistry & Cell Biology 112: 76–78. July 2019. doi:10.1016/j.biocel.2019.05.007. PMID 31085331. 
  14. ↑ "Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues". Brain Structure & Function 226 (2): 481–499. March 2021. doi:10.1007/s00429-020-02194-4. PMID 33386994. 
  15. ↑ "Novel imaging and related techniques for studies of diseases of the central nervous system: a review". Cell and Tissue Research 380 (3): 415–424. June 2020. doi:10.1007/s00441-020-03183-z. PMID 32072308. 
  16. ↑ "Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow". Science Translational Medicine 9 (387): eaah6518. April 2017. doi:10.1126/scitranslmed.aah6518. PMID 28446689. 
  17. ↑ "Phenotyping Intact Mouse Bones Using Bone CLARITY". Skeletal Development and Repair. Methods in Molecular Biology. 2230. New York, NY: Springer US. 2021. pp. 217–230. doi:10.1007/978-1-0716-1028-2_13. ISBN 978-1-0716-1028-2. 
  18. ↑ 18.0 18.1 "Imaging of the muscle and bone from benchtop to bedside". European Review for Medical and Pharmacological Sciences 24 (6): 3254–3266. March 2020. doi:10.26355/eurrev_202003_20693. PMID 32271443. 
  19. ↑ "Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method". Neurophotonics 7 (1): 015003. January 2020. doi:10.1117/1.NPh.7.1.015003. PMID 32016132. 
  20. ↑ "3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls". Scientific Reports 10 (1): 14276. August 2020. doi:10.1038/s41598-020-71174-9. PMID 32868776. Bibcode: 2020NatSR..1014276O. 
  21. ↑ "Cellular maps of gastrointestinal organs: getting the most from tissue clearing". American Journal of Physiology. Gastrointestinal and Liver Physiology 319 (1): G1–G10. July 2020. doi:10.1152/ajpgi.00075.2020. PMID 32421359. 
  22. ↑ "Imaging the developing human external and internal urogenital organs with light sheet fluorescence microscopy". Differentiation; Research in Biological Diversity 111: 12–21. 2020-01-01. doi:10.1016/j.diff.2019.09.006. PMID 31634681. https://escholarship.org/uc/item/8d39b32q. 
  23. ↑ "3D imaging of human epidermis micromorphology by combining fluorescent dye, optical clearing and confocal microscopy". Skin Research and Technology 25 (5): 735–742. September 2019. doi:10.1111/srt.12710. PMID 31074525. 
  24. ↑ "Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity" (in English). Nature Biomedical Engineering 1 (10): 796–806. October 2017. doi:10.1038/s41551-017-0139-0. PMID 31015588. 
  25. ↑ "Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies" (in English). Nature Biomedical Engineering 4 (9): 875–888. September 2020. doi:10.1038/s41551-020-0576-z. PMID 32601394. 



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