Tissue clearing explained

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

History

In the early 1900s, Werner Spalteholz developed a technique that allowed the clarification of large tissues,[7] [8] using Wintergrünöl (methyl salicylate) and benzyl benzoate.[9] Over the next hundred years, various scientists introduced their own variations on Spalteholz's technique. Tuchin et al. introduced TOC in 1997, adding a new branch of tissue clearing that was hydrophilic instead of hydrophobic like Spalteholz's technique.[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. Hama et al. developed another hydrophilic approach, Scale, in 2011. 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. A year later, in 2013, Chung et al. developed CLARITY, the first approach to use hydrogel monomers to clear tissue.

Principles

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

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. These include hydrophobic clearing methods, which may also be known as organic, solvent-based, organic solvent-based,[11] [12] or dehydration[13] clearing methods; hydrophilic clearing methods, which may also be known as aqueous-based or water-based methods, and may be further sub-categorized into simple immersion and hyperhydration (also called delipidation/ hydration); and hydrogel-based clearing methods, which may also be known as detergent or hydrogel embedding methods. Tissue-expansion clearing methods use hydrogel, and may be included under hydrogel-based clearing or as their own category.

Methods

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

Labeling

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

Imaging

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

Data

Imaging cleared tissues generates massive volumes of complex data, which requires powerful computational hardware and software to store, process, analyze, and visualize. A single mouse brain can generate terabytes of data. 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.

Applications

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

Notes and References

  1. Zhao J, Lai HM, Qi Y, He D, Sun H . Current Status of Tissue Clearing and the Path Forward in Neuroscience . ACS Chemical Neuroscience . 12 . 1 . 5–29 . January 2021 . 33326739 . 10.1021/acschemneuro.0c00563 . 229300600 .
  2. Vigouroux RJ, Belle M, Chédotal A . Neuroscience in the third dimension: shedding new light on the brain with tissue clearing . Molecular Brain . 10 . 1 . 33 . July 2017 . 28728585 . 5520295 . 10.1186/s13041-017-0314-y . free .
  3. Porter DD, Morton PD . Clearing techniques for visualizing the nervous system in development, injury, and disease . Journal of Neuroscience Methods . 334 . 108594 . January 2020 . 31945400 . 10.1016/j.jneumeth.2020.108594 . 210430342 . 10674098 .
  4. Tian T, Li X . Applications of tissue clearing in the spinal cord . The European Journal of Neuroscience . 52 . 9 . 4019–4036 . November 2020 . 32794596 . 10.1111/ejn.14938 . 221121163 .
  5. Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, Keller PJ . Tissue clearing and its applications in neuroscience . Nature Reviews. Neuroscience . 21 . 2 . 61–79 . February 2020 . 31896771 . 10.1038/s41583-019-0250-1. 8121164 . 209528204 .
  6. Gómez-Gaviro MV, Sanderson D, Ripoll J, Desco M . Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease . English . iScience . 23 . 8 . 101432 . August 2020 . 32805648 . 7452225 . 10.1016/j.isci.2020.101432 . 2020iSci...23j1432G .
  7. Ueda HR, Dodt HU, Osten P, Economo MN, Chandrashekar J, Keller PJ . Whole-Brain Profiling of Cells and Circuits in Mammals by Tissue Clearing and Light-Sheet Microscopy . Neuron . 106 . 3 . 369–387 . May 2020 . 32380050 . 7213014 . 10.1016/j.neuron.2020.03.004 .
  8. Azaripour A, Lagerweij T, Scharfbillig C, Jadczak AE, Willershausen B, Van Noorden CJ . 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 . 27142295 . 10.1016/j.proghi.2016.04.001 . free .
  9. Book: Spalteholz W . Über das Durchsichtigmachen von menschlichen und tierischen Präparaten und seine theoretischen Bedingungen, nebst Anhang: Über Knochenfärbung. S. Hirzel. 1914. Leipzig.
  10. Tuchin VV, Maksimova IL, Zimnyakov DA, Kon IL, Mavlyutov AH, Mishin AA . Light propagation in tissues with controlled optical properties . Journal of Biomedical Optics . 2 . 4 . 401–17 . October 1997 . 23014964 . 10.1117/12.281502 . 1997JBO.....2..401T . free .
  11. Tian T, Yang Z, Li X . Tissue clearing technique: Recent progress and biomedical applications . Journal of Anatomy . 238 . 2 . 489–507 . February 2021 . 32939792 . 7812135 . 10.1111/joa.13309 .
  12. Jing D, Yi Y, Luo W, Zhang S, Yuan Q, Wang J, Lachika E, Zhao Z, Zhao H . 6 . Tissue Clearing and Its Application to Bone and Dental Tissues . Journal of Dental Research . 98 . 6 . 621–631 . June 2019 . 31009584 . 6535919 . 10.1177/0022034519844510 .
  13. Watson AM, Watkins SC . Massive volumetric imaging of cleared tissue: The necessary tools to be successful . The International Journal of Biochemistry & Cell Biology . 112 . 76–78 . July 2019 . 31085331 . 10.1016/j.biocel.2019.05.007 . 155088859 .
  14. Kumar V, Krolewski DM, Hebda-Bauer EK, Parsegian A, Martin B, Foltz M, Akil H, Watson SJ . 6 . 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 . 33386994 . 7962668 . 10.1007/s00429-020-02194-4 .
  15. Dai Z, Sun Y, Zhao X, Pu X . 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 . 32072308 . 10.1007/s00441-020-03183-z . 211170939 .
  16. Greenbaum A, Chan KY, Dobreva T, Brown D, Balani DH, Boyce R, Kronenberg HM, McBride HJ, Gradinaru V . 6 . Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow . Science Translational Medicine . 9 . 387 . eaah6518 . April 2017 . 28446689 . 10.1126/scitranslmed.aah6518 . 8799170 .
  17. Book: Treweek JB, Beres A, Johnson N, Greenbaum A . Skeletal Development and Repair . Phenotyping Intact Mouse Bones Using Bone CLARITY . Methods in Molecular Biology . 2230 . 217–230 . 2021 . 33197017 . 10.1007/978-1-0716-1028-2_13 . Springer US . 978-1-0716-1028-2 . 226988513 . New York, NY . Hilton MJ .
  18. Wang HM, Khoradmehr A, Tamadon A, Velez E, Nabipour I, Jokar N, Assadi M, Gholamrezanezhad A . 6 . Imaging of the muscle and bone from benchtop to bedside . European Review for Medical and Pharmacological Sciences . 24 . 6 . 3254–3266 . March 2020 . 32271443 . 10.26355/eurrev_202003_20693 . 215602325 .
  19. Li Y, Xu J, Zhu J, Yu T, Zhu D . Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method . Neurophotonics . 7 . 1 . 015003 . January 2020 . 32016132 . 6977403 . 10.1117/1.NPh.7.1.015003 .
  20. Olianti C, Costantini I, Giardini F, Lazzeri E, Crocini C, Ferrantini C, Pavone FS, Camici PG, Sacconi L . 6 . 3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls . Scientific Reports . 10 . 1 . 14276 . August 2020 . 32868776 . 7459314 . 10.1038/s41598-020-71174-9 . 2020NatSR..1014276O .
  21. Liu CY, Polk DB . 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 . 32421359 . 7468759 . 10.1152/ajpgi.00075.2020 .
  22. Isaacson D, McCreedy D, Calvert M, Shen J, Sinclair A, Cao M, Li Y, McDevitt T, Cunha G, Baskin L . 6 . 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 . 31634681 . 10.1016/j.diff.2019.09.006 . 204833112 .
  23. Fernandez E, Marull-Tufeu S . 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 . 31074525 . 10.1111/srt.12710 . 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 . Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity . English . Nature Biomedical Engineering . 1 . 10 . 796–806 . October 2017 . 31015588 . 10.1038/s41551-017-0139-0 . 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 . Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies . English . Nature Biomedical Engineering . 4 . 9 . 875–888 . September 2020 . 32601394 . 10.1038/s41551-020-0576-z . 256704785 .

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