Medicine:Tissue clearing

From HandWiki

Tissue clearing refers to a group of chemical techniques used to turn tissues transparent.[1][2][3] By turning tissues transparent to certain wavelengths of light, it allows one to gain optical access to a tissue.[1] That is, light can pass into and out of the cleared tissue freely, allowing one to see the structures deep within the tissue without physically cutting it open. 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 useful only combined with one or more fluorescent labeling techniques such as immunolabeling 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] It is one of the more efficient ways to perform three-dimensional histology.

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] Various scientists then introduced their own variations on Spalteholz's technique.[8] Tuchin et al. introduced tissue optical clearing (TOC) in 1997, adding a new branch of tissue clearing that was hydrophilic instead of hydrophobic like Spalteholz's technique.[1][10] In the 1980s, Andrew Murray & Marc Kirschner developed a two-step process, wherein tissues were first dehydrated with alcohol and subsequently made transparent by immersion in a mixture of benzyl alcohol and benzyl benzoate (BABB), a technique they coupled with light sheet fluorescence microscopy,[11][2][3] which remains the method with the highest clearing efficacy to date, regardless any tissue pre-processing step.[12] In the most extreme case, it allows the clearing of a whole mouse of even a whole human brain.[13]

In 2024, Hong, Brongersma, and Ou reported that applying high concentrations of the food dye tartrazine could transiently and reversibly increase the optical transparency of certain biological tissues, including the skin, in live mice.[14][15][16] The authors attributed this effect to tartrazine's strong absorption in the blue region of the visible spectrum and to refractive index modulation at longer wavelengths, consistent with the Kramers–Kronig relations. Following publication, the findings have been independently reproduced and extended by multiple laboratories in several subsequent studies.[17][18][19][20] Specifically, this in vivo optical clearing approach has been applied by multiple independent laboratories to enhance imaging depth in modalities such as optical coherence tomography and photoacoustic imaging.[17][20][21][22][23][24] In 2025, Valery V. Tuchin, a pioneer in hydrophilic tissue clearing, demonstrated tartrazine can make the skull more transparent in live mice, enabling transcranial laser speckle imaging of cortical blood flow in real time.[18] In addition, a number of other labs have demonstrated the utility of tartrazine to enable deep-tissue Raman sensing[25] and fluorescence lifetime imaging.[26] In addition to tartrazine, several other absorbing dye molecules, including the FDA-approved contrast agents fluorescein and indocyanine green, have also been repurposed to function as in vivo optical clearing agents.[27][28][29] This observation suggests that the underlying physical principle of dye-enabled optical clearing is not limited to a single molecule and that multiple dye molecules may be repurposed as tissue clearing agents.

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 there are multiple class names for tissue-clearing methods, they are all classified based on the final state of the tissue by the end of the clearing method.[1] These include hydrophobic clearing methods,[1][2][6] which may also be known as organic,[3] solvent-based,[4][5] organic solvent-based,[30][31] or dehydration[32] clearing methods; hydrophilic clearing methods,[1][2][6] which may also be known as aqueous-based[5][30] or water-based[32] methods, and hydrogel-based clearing methods.[2][1]

Labeling

Tissue clearing methods have varying compatibility with different methods of fluorescent labeling.[1][5][6] Some are better suited to genetic labelling by endogenously expressed fluorescent protein,[1][5] while others externally delivered probes as immunolabeling and chemical dye labeling.[1][5] The latter is more general and applicable to all tissues, notably human tissues, but the penetration of the probes becomes a critical problem.[33]

Imaging

After clearing and labeling, tissues are typically imaged using confocal microscopy,[30][31][32] two-photon microscopy,[1][5][30] or one of the many variants of light-sheet fluorescence microscopy.[7][30][31] Other less commonly used methods include optical projection tomography[1][5] and stimulated Raman scattering.[5][7][30] As long as the tissue allows for the unobstructed passing of light, the optical resolution is fundamentally limited by Abbe diffraction limit. The compatibility of any tissue clearing method with any microscopy system is, therefore, configurational rather than optical.

Data

Tissue clearing is one of the more efficient ways to facilitate 3D imaging of tissues, and hence generates massive volumes of complex data, which requires powerful computational hardware and software to store, process, analyze, and visualize.[1][6][32] A single mouse brain can generate terabytes of data.[2][6][32] 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][30][31]

Applications

Tissue clearing has been applied to the nervous system,[1][2][3][4][5][6][7][30][34][35] bones (including teeth),[7][30][31][36][37][38] skeletal muscles,[7][38][39] hearts and vasculature,[7][30][40] gastrointestinal organs,[7][41] urogenital organs,[7][30][42] skin,[7][43] lymph nodes,[7] mammary glands,[7] lungs,[7] eyes,[7] tumors,[7][30] and adipose tissues.[7][30] 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.[44][45] For some techniques, bone tissue must be decalcified to remove light-scattering hydroxyapatite crystals, leaving behind a protein matrix suitable for clearing.[46][47]

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 "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. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 "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 "Neuroscience in the third dimension: shedding new light on the brain with tissue clearing". Molecular Brain 10 (1). July 2017. doi:10.1186/s13041-017-0314-y. PMID 28728585. 
  4. ↑ 4.0 4.1 4.2 4.3 4.4 4.5 "Clearing techniques for visualizing the nervous system in development, injury, and disease". Journal of Neuroscience Methods 334. 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 "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). August 2020. doi:10.1016/j.isci.2020.101432. PMID 32805648. Bibcode: 2020iSci...23j1432G. 
  8. ↑ 8.0 8.1 "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. ↑ Dent; Polson; Klymkowsky (1989). "A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus". Development 105 (1): 61–74. doi:10.1242/dev.105.1.61. PMID 2806118. 
  12. ↑ Pan, Chenchen; Cai, Ruiyao; Quacquarelli, Francesca Paola; Ghasemigharagoz, Alireza; Lourbopoulos, Athanasios; Matryba, Paweł; Plesnila, Nikolaus; Dichgans, Martin et al. (2016). "Shrinkage-mediated imaging of entire organs and organisms using uDISCO". Nature Methods 13 (10): 859–867. doi:10.1038/nmeth.3964. PMID 27548807. 
  13. ↑ Zhao, Shan; Mihail Ivilinov, Todorov; Ruiyao, Cai; Rami, AI -Maskari; Hanno, Steinke; Elisabeth, Kemter; Hongcheng, Mai; Zhouyi, Rong et al. (2020). "Cellular and Molecular Probing of Intact Human Organs". Cell 2020 (4): 796–812.e19. doi:10.1016/j.cell.2020.01.030. PMID 32059778. 
  14. ↑ Ou, Zihao; Duh, Yi-Shiou; Rommelfanger, Nicholas J.; Keck, Carl H. C.; Jiang, Shan; Brinson, Kenneth; Zhao, Su; Schmidt, Elizabeth L. et al. (2024-09-06). "Achieving optical transparency in live animals with absorbing molecules". Science 385 (6713). doi:10.1126/science.adm6869. PMID 39236186. Bibcode: 2024Sci...385m6869O. 
  15. ↑ Keck, Carl H. C.; Schmidt, Elizabeth Lea; Zhao, Su; Liu, Zhongyu; Zhang, Ling-Yi; Cui, Miao; Chen, Xiaoyu; Wang, Chonghe et al. (2025-05-13). "Achieving transient and reversible optical transparency in live mice with tartrazine" (in en). Nature Protocols 21 (1): 319–346. doi:10.1038/s41596-025-01187-z. ISSN 1750-2799. PMID 40360854. https://www.nature.com/articles/s41596-025-01187-z. 
  16. ↑ Shabbir, Muhammad Waqas; Asante-Asare, David; Phillips, Matthew; Ou, Zihao (2025-07-11). "Transient Optical Clearing Using Absorbing Molecules for Ex Vivo and In Vivo Imaging" (in en). Journal of Visualized Experiments (221). doi:10.3791/68629. ISSN 1940-087X. PMID 40720350. https://app.jove.com/t/68629/transient-optical-clearing-using-absorbing-molecules-for-ex-vivo-vivo. 
  17. ↑ 17.0 17.1 Miller, David A.; Xu, Yirui; Highland, Robert; Nguyen, Van Tu; Brown, William J.; Hong, Guosong; Yao, Junjie; Wax, Adam (2025-01-20). "Enhanced penetration depth in optical coherence tomography and photoacoustic microscopy in vivo enabled by absorbing dye molecules" (in en). Optica 12 (1): 24–30. doi:10.1364/OPTICA.546779. ISSN 2334-2536. PMID 41625956. PMC 12858289. Bibcode: 2025Optic..12...24M. https://opg.optica.org/abstract.cfm?URI=optica-12-1-24. 
  18. ↑ 18.0 18.1 Surkov, Y.; Timoshina, P.; Uvakin, I.; Shushunova, N.; Konovalov, A.; Kozlov, I.; Piavchenko, G.; Telyshev, D. et al. (2025-10-16). "Computer-guided optical clearing for transcranial laser speckle imaging of cortical blood flow through synergistic tartrazine-induced cranial bone transparency". Journal of Innovative Optical Health Sciences. doi:10.1142/S1793545825400024. ISSN 1793-5458. https://www.worldscientific.com/doi/10.1142/S1793545825400024. 
  19. ↑ Tie, Xin; Sun, Ting; Xiao, Guixiu; Zhao, Yanjie; Su, Jing; Xie, Xiaoqi; Yin, Wanhong (2024-11-01). "Absorbing molecules make both abdomen and back transparent in live mice". bioRxiv 10.1101/2024.10.28.620537.
  20. ↑ 20.0 20.1 Zuo, Tianxiang; Tao, Chao; Liu, Xiaojun (2025-04-01). "Absorbing molecules as optical clearing agents improve the resolution and sensitivity of photoacoustic microscopy" (in en). Optics Letters 50 (7): 2282–2285. doi:10.1364/OL.555723. ISSN 0146-9592. PMID 40167701. Bibcode: 2025OptL...50.2282Z. https://opg.optica.org/abstract.cfm?URI=ol-50-7-2282. 
  21. ↑ Jia, Conger; Zhang, Zhiling; Shen, Yuecheng; Hou, Wanli; Zhao, Jiayu; Luo, Jiawei; Chen, Haoran; Qi, Dalong et al. (2025-06-01). "Tartrazine-enabled optical clearing for in vivo optical resolution photoacoustic microscopy" (in en). Biomedical Optics Express 16 (6): 2504–2515. doi:10.1364/BOE.565643. ISSN 2156-7085. PMID 40677379. PMC 12265485. https://opg.optica.org/abstract.cfm?URI=boe-16-6-2504. 
  22. ↑ Xu, Maoyuan; Yang, Bingqian; Song, Shen; Xu, Tianpeng; Yao, Jinyu; Liu, Yuehao; Cui, Yaoyao; Zhang, Yachao (2025-10-01). "Multi-wavelength photoacoustic microscopy enhanced by the high-sensitivity probe and reversible tissue transparent molecules" (in en). Photonics Research 13 (10): 2757. doi:10.1364/PRJ.565972. ISSN 2327-9125. https://opg.optica.org/abstract.cfm?URI=prj-13-10-2757. 
  23. ↑ Liang, Yilin; Meng, Xiaochen; Wang, Chongyang; Ma, Jiawei; Zhang, Xuanye; Fan, Fan; Zhu, Jiang (September 2025). "Optical Coherence Tomography and Angiography Image Enhancement Using Optical Clearing Agent Tartrazine" (in en). Journal of Biophotonics 19 (1). doi:10.1002/jbio.202500297. ISSN 1864-063X. PMID 40890078. https://onlinelibrary.wiley.com/doi/10.1002/jbio.202500297. 
  24. ↑ Narawane, Amit; Trout, Robert; Viehland, Christian; Kuo, Anthony N.; Vajzovic, Lejla; Dhalla, Al-Hafeez; Toth, Cynthia A. (2024-12-09). "Optical clearing with tartrazine enables deep transscleral imaging with optical coherence tomography". Journal of Biomedical Optics 29 (12). doi:10.1117/1.JBO.29.12.120501. ISSN 1083-3668. PMID 39669907. Bibcode: 2024JBO....29l0501N. 
  25. ↑ Lee, Michael Ka Ho; Mizushima, Kenta; Zheng, Peng; Tanwar, Swati; Gupta, Anoushka; Fujita, Katsumasa; Barman, Ishan (2025-10-24). "Spectrally Silent and Optically Transparent: Clear-SiR for Deep Raman Biomolecular Sensing". ACS Sensors 10 (10): 7702–7711. doi:10.1021/acssensors.5c02046. PMID 41024637. Bibcode: 2025ACSSe..10.7702L. 
  26. ↑ Yuan, Nanxue; Ragab, Saif; Chavez, Luis; Pandey, Vikas; Intes, Xavier (2025-12-15). "Evaluating tartrazine as an optical clearing agent for fluorescence lifetime imaging" (in en). Optics Letters 50 (24): 7588–7591. doi:10.1364/OL.579040. ISSN 0146-9592. PMID 41396940. Bibcode: 2025OptL...50.7588Y. https://opg.optica.org/abstract.cfm?URI=ol-50-24-7588. 
  27. ↑ Trout, Robert M; Narawane, Amit; Viehland, Christian; Ownagh, Vahid; Draelos, Mark; Dhalla, Al-Hafeez; Kuo, Anthony; Toth, Cynthia (2026). "Optical Coherence Tomography with Fluorescein Optical Clearing for Transscleral Image Guidance" (in en). International Journal of Translational Medicine 6: 7. doi:10.3390/ijtm6010007. 
  28. ↑ Lu, Kechao; Xu, Yirui; Miller, David A.; Wang, Wan; Gupta, Deven; Yao, Junjie; Wax, Adam (2025-09-01). "Indocyanine green (ICG) enhances penetration of 1300 nm optical coherence tomography imaging for in vivo murine skin" (in en). Optics Letters 50 (17): 5226–5229. doi:10.1364/OL.569764. ISSN 0146-9592. PMID 40882048. PMC 12848760. Bibcode: 2025OptL...50.5226L. https://opg.optica.org/abstract.cfm?URI=ol-50-17-5226. 
  29. ↑ Keck, Carl H. C.; Schmidt, Elizabeth L.; Roth, Richard H.; Floyd, Brendan M.; Tsai, Andy P.; Garcia, Hassler B.; Cui, Miao; Chen, Xiaoyu et al. (2025-09-02). "Color-neutral and reversible tissue transparency enables longitudinal deep-tissue imaging in live mice". Proceedings of the National Academy of Sciences 122 (35). doi:10.1073/pnas.2504264122. PMID 40857313. Bibcode: 2025PNAS..12204264K. 
  30. ↑ 30.00 30.01 30.02 30.03 30.04 30.05 30.06 30.07 30.08 30.09 30.10 30.11 30.12 "Tissue clearing technique: Recent progress and biomedical applications". Journal of Anatomy 238 (2): 489–507. February 2021. doi:10.1111/joa.13309. PMID 32939792. 
  31. ↑ 31.0 31.1 31.2 31.3 31.4 "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. 
  32. ↑ 32.0 32.1 32.2 32.3 32.4 "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. 
  33. ↑ Yau, Chun Ngo; Hei Ming, Lai (2023). "Principles of deep immunohistochemistry for 3D histology". Cell Reports Methods 3 (5). doi:10.1016/j.crmeth.2023.100458. PMID 37323568. 
  34. ↑ "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. 
  35. ↑ "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. 
  36. ↑ "Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow". Science Translational Medicine 9 (387). April 2017. doi:10.1126/scitranslmed.aah6518. PMID 28446689. https://resolver.caltech.edu/CaltechAUTHORS:20161221-135116035. 
  37. ↑ "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. 
  38. ↑ 38.0 38.1 "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. 
  39. ↑ "Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method". Neurophotonics 7 (1). January 2020. doi:10.1117/1.NPh.7.1.015003. PMID 32016132. 
  40. ↑ "3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls". Scientific Reports 10 (1). August 2020. doi:10.1038/s41598-020-71174-9. PMID 32868776. Bibcode: 2020NatSR..1014276O. 
  41. ↑ "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. 
  42. ↑ "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. 
  43. ↑ "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. 
  44. ↑ "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. 
  45. ↑ "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. 
  46. ↑ "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. 
  47. ↑ "Neuroscience in the third dimension: shedding new light on the brain with tissue clearing". Molecular Brain 10 (1). July 2017. doi:10.1186/s13041-017-0314-y. PMID 28728585. 



Retrieved from "https://handwiki.org/wiki/index.php?title=Medicine:Tissue_clearing&oldid=4305606"