LABTips: How to Select a Tissue Clearing Method

The natural opacity of most biological tissues typically limits the resolution of microscopic investigations using visible light at depths greater than 100 µm. Tissue clearing is the chemical process of rendering tissues transparent in order to improve resolution and imaging depth in thicker tissues, which can enable better 3D visualizations than can be achieved using other approaches such as slicing and digitally reconstructing thinner sections of tissue. There are several different categories of tissue clearing strategies, and dozens of specific protocols that have been published, all with the end goal of bringing tissue components to a common refractive index (RI). If you are new to tissue clearing, figuring out where to start and which clearing techniques to try can be daunting; the following tips can help you narrow down your options to find the best technique for your application.

1. Investigate the principles behind different categories of clearing methods

The objective of tissue clearing is to reduce light scattering, which is caused by a mixture of different components with different refractive indices within tissue (especially lipids, proteins and water), as well as light absorption from components such as hemoglobin and melanin. Chemical clearing processes generally include removal of some components (decolorization, dehydration and/or delipidation) and immersion of the sample in a solution that homogenizes the RI of the remaining tissue (RI matching). While there is a vast array of specific, published tissue clearing techniques – and even more optimizations and derivatives of these established methods – most clearing protocols fall within a set of broader categories based on the principles and steps used to render tissues transparent. Three main categories are organic solvent-based clearing, aqueous-based clearing and hydrogel embedding.^1,2

Organic solvent tissue clearing methods involve both dehydration and delipidation of the sample followed by RI matching of the remaining components via immersion in high RI organic solvents. Examples include benzyl alcohol:benzyl benzoate (BABB) and “3D imaging of solvent-cleared organs” (3DISCO), which uses tetrahydrofuran (THF) for dehydration, dichloromethane (DCM) for delipidation and dibenzyl ether (DBE) for RI matching. These techniques bring the tissue to a final RI of about 1.55 and 1.56, respectively.

Aqueous-based clearing, by contrast, does not require dehydration and uses aqueous clearing agents instead of hydrophobic organic solvents; this can include simple immersion in a high RI aqueous solution or hyperhydration, which lowers the RI of proteins. This latter technique can also include lipid removal to further improve transparency. Clear^T is an example of a simple immersion method that involves immersion in graded concentrations of formamide in phosphate buffered saline (PBS), which brings the tissue to a final RI of about 1.45.³ Scale A2 is a hyperhydration solution containing urea, glycerol and Triton X-100 and lowers the RI to 1.38.

Hydrogel embedding is a specialized technique in which proteins are cross linked with a synthetic gel, which serves to immobilize them and preserve their structure while allowing for thorough removal of lipids prior to RI matching. These methods may also be paired with electrophoretic tissue clearing (ETC) for more rapid clearing and immunolabeling.^1,2 CLARITY (“Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel”) was the first such example using acrylamide-based hydrogel and ETC with a strong sodium dodecyl sulfate (SDS) detergent for lipid removal; the final RI of CLARITY-cleared tissues is about 1.45.

Understanding how different clearing techniques are classified and the steps they involve can help you consider factors such as solvent safety and cost, the cost and complexity of required equipment, clearing speed and quality and compatible labeling strategies.

2. Assess compatibility with labeling methods

One of the most important things to know about any tissue clearing method is its compatibility with fluorescent labeling methods, including fluorescent proteins, antibodies and lipid staining. It goes without saying that any clearing method that includes delipidation is incompatible with direct lipid staining. If lipid staining is critical for your research, the selection of clearing methods narrows significantly as lipids are a major cause of light scatter and a majority of staining methods aim to remove them. Simple immersion methods like SeeDB,⁵ which uses an aqueous solution of fructose and thioglycerol to clear tissue while preserving lipophilic neuronal tracers, and certain hyperhydration methods like the sorbitol-based ScaleS,⁶ are available to increase transparency without removing lipids.

Aqueous-based and hydrogel-based clearing methods are advantageous for preserving fluorescent proteins, as quenching of fluorescent proteins due to dehydration is a major drawback of organic solvent-based methods. However, there are a few organic solvent methods that have been optimized to extend fluorescent protein preservation from hours or days to months and even years, including the polyethylene glycol (PEG)-associated solvent system (PEGASOS), and the 3DISCO derivatives uDISCO and sDISCO.¹

For studies that require immunostaining, there are two factors to consider – compatibility with antibody labels and clearing speed. The latter factor is crucial when taking into account that adding more days or weeks onto already-lengthy immunostaining incubation times could render the preparation procedure prohibitively time-consuming.⁴ Aqueous-based methods, while amenable to immunostaining, face the problem of long immersion times, especially for larger samples. Hydrogel methods offer the advantage of enabling multiple rounds of de-staining and re-labeling, but can also be time consuming, especially without the use of ETC, which requires specialized equipment. One method, called PRESTO (“pressure related efficient and stable transfer of macromolecules into organs”) offers improvements in labeling speed and depth using either centrifugal force or a syringe pump system to facilitate expedited antibody delivery.⁷ Organic solvent methods are especially rapid and useful in combination with immunostaining, with iDISCO being one method specific optimized for immunolabeling of large, cleared samples.⁸

3. Consider the size of the sample

Tissue clearing is used to improve imaging of samples ranging in thickness from fractions of a millimeter to several centimeters.⁴ The size of the sample will largely affect the time needed to complete the clearing process, and some protocols are specifically optimized for especially large samples ranging from whole organs to whole organisms.

For relatively small samples (less than 1-2 mm thick), one can take advantage of the simplicity and safety of aqueous-based methods, while avoiding the fluorescent protein quenching and tissue shrinkage that can occur when using organic solvent methods. As sample thickness increases, however, immersion times for these methods can extend to weeks or even months. Organic solvent methods offer the highest clearing speeds, making them more suitable for large samples – for example, 3DISCO can clear a whole adult mouse brain in just three days, and uDISCO can clear a whole mouse body (without skin) in around the same time.¹

Hydrogel methods using passive diffusion only will also require long incubation times (up to weeks) for larger tissues. While active methods (using ETC) can shorten the time to just hours, they can also cause damage to tissues due to the use of electric fields and heating up of the electrophoresis chamber.⁹ The use of stochastic electrotransport can improve speed with less tissue damage, and there are additional ECT-independent hydrogel methods that optimize clearing speed, such as SWITCH (“system-wide control of interaction time and kinetics of chemicals”), which enables the use of high temperatures to accelerate clearing while securing tissue architecture and native biomolecules. This technique enables the clearing of a whole adult mouse brain within 4 days at temperatures of 80°C.¹⁰

Whole-body clearing requires a method that offers both the speed to clear a large sample and the versatility to clear multiple tissue types. PEGASOS is an example of an organic solvent method that can clear both soft and hard tissues, including bone, in about 12 days (from fixation to clearing completion) for a whole adult mouse body.¹⁰ As mentioned, uDISCO can also be used for rapid whole body clearing, and also dramatically shrinks samples in a manner that is advantageous for scanning-based imaging modalities.¹¹ With a wide range of specialized protocols available for a range of tissue types, organs and organisms, searching the research literature for successful applications on similar samples is a helpful starting point for developing a method that supports your specific research objectives.

References

1. Tian, T, Yang, Z, Li, X, et al. Tissue clearing technique: Recent progress and biomedical applications. J Anat. 2021; 238: 489– 507. https://doi.org/10.1111/joa.13309
2. "Seeing clearly yet?: An overview of tissue clearing," Blog Post by Massimo Onesto, Biodock (2022). https://blog.biodock.ai/seeing-clearly-yet-an-overview-of-tissue-clearing-for-deep-specimen-imaging/
3. Paysan, J. The Art of Tissue Clearing [Online]; Essential Knowledge Briefings; John Wiley & Sons Ltd: Chichester, West Sussex, UK, 2021. https://www.essentialknowledgebriefings.com/downloads/the-art-of-tissue-clearing/# (accessed Sept 14, 2022). 
4. Ariel, P. A Beginner’s Guide to Tissue Clearing. The International Journal of Biochemistry & Cell Biology 2017, 84, 35–39. https://doi.org/10.1016/j.biocel.2016.12.009.
5. Ke, MT., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16, 1154–1161 (2013). https://doi.org/10.1038/nn.3447
6. Hama, H., Hioki, H., Namiki, K. et al. ScaleS: an optical clearing palette for biological imaging. Nat Neurosci 18, 1518–1529 (2015). https://doi.org/10.1038/nn.4107
7. Lee, E., Choi, J., Jo, Y. et al. ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep 6, 18631 (2016). https://doi.org/10.1038/srep18631
8. Renier, N.; Wu, Z.; Simon, David J.; Yang, J.; Ariel, P.; Tessier-Lavigne, M. IDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 2014, 159 (4), 896–910. https://doi.org/10.1016/j.cell.2014.10.010.
9. Guo, Z.; Zheng, Y.; Zhang, Y. CLARITY Techniques Based Tissue Clearing: Types and Differences. Folia Morphologica 2022, 81 (1), 1–12. https://doi.org/10.5603/fm.a2021.0012.
10. Murray, E.; Cho, J. H.; Goodwin, D.; Ku, T.; Swaney, J.; Kim, S.-Y.; Choi, H.; Park, Y.-G.; Park, J.-Y.; Hubbert, A.; McCue, M.; Vassallo, S.; Bakh, N.; Frosch, M. P.; Wedeen, V. J.; Seung, H. S.; Chung, K. Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems. Cell 2015, 163 (6), 1500–1514. https://doi.org/10.1016/j.cell.2015.11.025.
11. Evans, C. L. Seeing Is Believing: Tissue Clearing Makes See-through Rodents. Science Translational Medicine 2016, 8 (354), 354ec138–354ec138. https://doi.org/10.1126/scitranslmed.aah6000.