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[edit]

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[edit]

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[edit]

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/ hydration[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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

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[edit]

  1. ^ a b c d e f g h i j k l m n o p q r s t u v w x y z Zhao J, Lai HM, Qi Y, He D, Sun H (January 2021). "Current Status of Tissue Clearing and the Path Forward in Neuroscience". ACS Chemical Neuroscience. 12 (1): 5–29. doi:10.1021/acschemneuro.0c00563. PMID 33326739. S2CID 229300600.
  2. ^ a b c d e f g h i j k l m n o p q Ueda HR, Dodt HU, Osten P, Economo MN, Chandrashekar J, Keller PJ (May 2020). "Whole-Brain Profiling of Cells and Circuits in Mammals by Tissue Clearing and Light-Sheet Microscopy". Neuron. 106 (3): 369–387. doi:10.1016/j.neuron.2020.03.004. PMC 7213014. PMID 32380050.
  3. ^ a b c d e f g h Vigouroux RJ, Belle M, Chédotal A (July 2017). "Neuroscience in the third dimension: shedding new light on the brain with tissue clearing". Molecular Brain. 10 (1): 33. doi:10.1186/s13041-017-0314-y. PMC 5520295. PMID 28728585.
  4. ^ a b c d e f g h i j k l m n o Porter DD, Morton PD (January 2020). "Clearing techniques for visualizing the nervous system in development, injury, and disease". Journal of Neuroscience Methods. 334: 108594. doi:10.1016/j.jneumeth.2020.108594. PMC 10674098. PMID 31945400. S2CID 210430342.
  5. ^ a b c d e f g h i j k l m n o Tian T, Li X (November 2020). "Applications of tissue clearing in the spinal cord". The European Journal of Neuroscience. 52 (9): 4019–4036. doi:10.1111/ejn.14938. PMID 32794596. S2CID 221121163.
  6. ^ a b c d e f g h i Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, Keller PJ (February 2020). "Tissue clearing and its applications in neuroscience". Nature Reviews. Neuroscience. 21 (2): 61–79. doi:10.1038/s41583-019-0250-1. PMC 8121164. PMID 31896771. S2CID 209528204.
  7. ^ a b c d e f g h i j k l m n o p q Gómez-Gaviro MV, Sanderson D, Ripoll J, Desco M (August 2020). "Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease". iScience. 23 (8): 101432. Bibcode:2020iSci...23j1432G. doi:10.1016/j.isci.2020.101432. PMC 7452225. PMID 32805648.
  8. ^ a b c d e Azaripour A, Lagerweij T, Scharfbillig C, Jadczak AE, Willershausen B, Van Noorden CJ (August 2016). "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. doi:10.1016/j.proghi.2016.04.001. PMID 27142295.
  9. ^ Spalteholz W (1914). Über das Durchsichtigmachen von menschlichen und tierischen Präparaten und seine theoretischen Bedingungen, nebst Anhang: Über Knochenfärbung. Leipzig: S. Hirzel.
  10. ^ Tuchin VV, Maksimova IL, Zimnyakov DA, Kon IL, Mavlyutov AH, Mishin AA (October 1997). "Light propagation in tissues with controlled optical properties". Journal of Biomedical Optics. 2 (4): 401–17. Bibcode:1997JBO.....2..401T. doi:10.1117/12.281502. PMID 23014964.
  11. ^ a b c d e f g h i j k l m n Tian T, Yang Z, Li X (February 2021). "Tissue clearing technique: Recent progress and biomedical applications". Journal of Anatomy. 238 (2): 489–507. doi:10.1111/joa.13309. PMC 7812135. PMID 32939792.
  12. ^ a b c d e Jing D, Yi Y, Luo W, Zhang S, Yuan Q, Wang J, et al. (June 2019). "Tissue Clearing and Its Application to Bone and Dental Tissues". Journal of Dental Research. 98 (6): 621–631. doi:10.1177/0022034519844510. PMC 6535919. PMID 31009584.
  13. ^ a b c d e Watson AM, Watkins SC (July 2019). "Massive volumetric imaging of cleared tissue: The necessary tools to be successful". The International Journal of Biochemistry & Cell Biology. 112: 76–78. doi:10.1016/j.biocel.2019.05.007. PMID 31085331. S2CID 155088859.
  14. ^ Kumar V, Krolewski DM, Hebda-Bauer EK, Parsegian A, Martin B, Foltz M, et al. (March 2021). "Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues". Brain Structure & Function. 226 (2): 481–499. doi:10.1007/s00429-020-02194-4. PMC 7962668. PMID 33386994.
  15. ^ Dai Z, Sun Y, Zhao X, Pu X (June 2020). "Novel imaging and related techniques for studies of diseases of the central nervous system: a review". Cell and Tissue Research. 380 (3): 415–424. doi:10.1007/s00441-020-03183-z. PMID 32072308. S2CID 211170939.
  16. ^ Greenbaum A, Chan KY, Dobreva T, Brown D, Balani DH, Boyce R, et al. (April 2017). "Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow". Science Translational Medicine. 9 (387): eaah6518. doi:10.1126/scitranslmed.aah6518. PMID 28446689. S2CID 8799170.
  17. ^ Treweek JB, Beres A, Johnson N, Greenbaum A (2021). "Phenotyping Intact Mouse Bones Using Bone CLARITY". In Hilton MJ (ed.). Skeletal Development and Repair. Methods in Molecular Biology. Vol. 2230. New York, NY: Springer US. pp. 217–230. doi:10.1007/978-1-0716-1028-2_13. ISBN 978-1-0716-1028-2. PMID 33197017. S2CID 226988513.
  18. ^ a b Wang HM, Khoradmehr A, Tamadon A, Velez E, Nabipour I, Jokar N, et al. (March 2020). "Imaging of the muscle and bone from benchtop to bedside". European Review for Medical and Pharmacological Sciences. 24 (6): 3254–3266. doi:10.26355/eurrev_202003_20693. PMID 32271443. S2CID 215602325.
  19. ^ Li Y, Xu J, Zhu J, Yu T, Zhu D (January 2020). "Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method". Neurophotonics. 7 (1): 015003. doi:10.1117/1.NPh.7.1.015003. PMC 6977403. PMID 32016132.
  20. ^ Olianti C, Costantini I, Giardini F, Lazzeri E, Crocini C, Ferrantini C, et al. (August 2020). "3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls". Scientific Reports. 10 (1): 14276. Bibcode:2020NatSR..1014276O. doi:10.1038/s41598-020-71174-9. PMC 7459314. PMID 32868776.
  21. ^ Liu CY, Polk DB (July 2020). "Cellular maps of gastrointestinal organs: getting the most from tissue clearing". American Journal of Physiology. Gastrointestinal and Liver Physiology. 319 (1): G1–G10. doi:10.1152/ajpgi.00075.2020. PMC 7468759. PMID 32421359.
  22. ^ Isaacson D, McCreedy D, Calvert M, Shen J, Sinclair A, Cao M, et al. (2020-01-01). "Imaging the developing human external and internal urogenital organs with light sheet fluorescence microscopy". Differentiation; Research in Biological Diversity. 111: 12–21. doi:10.1016/j.diff.2019.09.006. PMID 31634681. S2CID 204833112.
  23. ^ Fernandez E, Marull-Tufeu S (September 2019). "3D imaging of human epidermis micromorphology by combining fluorescent dye, optical clearing and confocal microscopy". Skin Research and Technology. 25 (5): 735–742. doi:10.1111/srt.12710. PMID 31074525. S2CID 149445451.
  24. ^ Tanaka N, Kanatani S, Tomer R, Sahlgren C, Kronqvist P, Kaczynska D, Louhivuori L, Kis L, Lindh C, Mitura P, Stepulak A, Corvigno S, Hartman J, Micke P, Mezheyeuski A, Strell C, Carlson JW, Fernández Moro C, Dahlstrand H, Östman A, Matsumoto K, Wiklund P, Oya M, Miyakawa A, Deisseroth K, Uhlén P (October 2017). "Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity". Nature Biomedical Engineering. 1 (10): 796–806. doi:10.1038/s41551-017-0139-0. PMID 31015588. S2CID 256713371.
  25. ^ Tanaka N, Kanatani S, Kaczynska D, Fukumoto K, Louhivuori L, Mizutani T, Kopper O, Kronqvist P, Robertson S, Lindh C, Kis L, Pronk R, Niwa N, Matsumoto K, Oya M, Miyakawa A, Falk A, Hartman J, Sahlgren C, Clevers H, Uhlén P (September 2020). "Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies". Nature Biomedical Engineering. 4 (9): 875–888. doi:10.1038/s41551-020-0576-z. PMID 32601394. S2CID 256704785.
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