Tissue Clearing

Imaging long-range functional correlations and connections is of vital importance in biomedical research, especially in the central and peripheral nervous system. Unfortunately, biological samples absorb and scatter visible light, and it is not possible to image tissue sections ticker than 50 to 100 um under a regular microscope. Two-photon microscopy alleviates this problem by using higher-penetrating infra-red light, but it requires heavy equipment. Serial sectioning is also an alternative for imaging thick tissue specimens, but registration of serial sections is notoriously difficult. An easier option is to clear the tissue, thereby making it more transparent to visible light. Once tissue is cleared, the depth of microscopy imaging is only limited by the working distance of the objective (500 - 600 um with a Zeiss 25x LCI lens).

Tissue clearing improves the penetration of light by reducing the lipid and water content of the tissue while preserving proteins and nucleic acids. A variety clearing methods have been developed in recent years (1). The procedures typically includes 4 steps: tissue fixation, lipid extraction, antibody staining and refractive index (RI) matching. Tissue clearing methods can be classified into three major types:

  1. Hydrophobic tissue clearing uses aggressive organic solvents for tissue permeabilization and delipidation. Solvents are mixtures of benzyl alcohol and benzyl benzoate (BABB). Dibenzyl ether (DBE) is used for RI matching. These methods are quick and simple but may cause a loss of endogenous biomolecules, especially lipid-associated proteins. iDISCO (2) belongs to this type of methods.
  2. Hydrophilic tissue clearing uses detergents such as SDS (sodium dodecyl sulfate) or Triton X-100 for delipidation and permeabilization. Sucrose, sorbitol, urea, alone or in combination, are used for RI matching. These reagents are less toxic and endogenous biomolecules are normally well preserved, but these procedures take more time than the hydrophobic methods. The CUBIC protocols is one such technique (3,4).
  3. Hydrogel-based tissue clearing embeds the tissue in hydrogels matrixes such as acrylamide (CLARITY (5)) or polyepoxide (SHIELD (6)) before delipidation and permeabilization. Cross-links between the hydrogel and the tissue ensure the structures and organization of endogenous biomolecules are well preserved. These methods are time-consuming, complex protocols.

Tissue clearing is a complex field where the choice of method is determined by the tissue type, the goal of the experiment, the characteristics of the proteins or biomolecules of interest, and the imaging requirements. The MIC provides a high level of support for cleared sample preparation and imaging. Contact Dr. Ling Yi (yil@mail.nih.gov) for details.

Refer to caption.
Adult mouse brain cleared with CUBIC. Left to right: uncleared, CUBIC 3 weeks, CUBIC 4 weeks. Ling Yi, MIC, NICHD.


  1. Parra-Damas A, Saura CA. Tissue Clearing and Expansion Methods for Imaging Brain Pathology in Neurodegeneration: From Circuits to Synapses and Beyond. Front Neurosci. 2020 Oct 5;14:914. 
  2. Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P., and Tessier-Lavigne, M. (2014). iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910.
  3. Susaki, E. A., Tainaka, K., Perrin, D., Kishino, F., Tawara, T., Watanabe, T. M., et al. (2014). Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739.
  4. Murakami,T.C., Mano T., Saikawa S., Horiguchi S.A., et al (2018). A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.  Nature Neuroscience 21, 625–637.
  5. Chung, K., Wallace, J., Kim, S.-Y., Kalyanasundaram, S., Andalman, A. S., Davidson, T. J., et al. (2013). Structural and molecular interrogation of intact biological systems. Nature 497, 332–337.
  6. Park, Y.-G., Sohn, C. H., Chen, R., McCue, M., Yun, D. H., Drummond, G. T., et al. (2019). Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat. Biotechnol. 37, 73–83.

Additional information

CUBIC Tissue Clearing Protocol (PDF 171 KB)

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