Tissue clearing is an efficacious, new array of methods that transformed biological imaging. Tissue clearing is ultimately about making biological tissues transparent, while maintaining structure and molecular integrity. Its transparency is key to high-resolution, 3D imaging of tissues and organs, so that scientists can understand the architecture of tissues and discover how they function.

Decomposition of tissues varies from technique to technique through the years, each with its own advantages and limitations in relation to the tissue, research goals, and imaging requirements. In this article, we are going to classify and explore the most widely used tissue clearing techniques in detail – what they do, how they can be used, and their limitations.

Solvent-Based Tissue Clearing

Biological tissues are generally cleared using solvents, which disperse light on them. These solvents tend to dissolve water from the tissue into something that's glassy.

Figure 1. Different steps for the four classifications of tissue-clearing techniques. (Zhan YJ, et al.; 2023)

Cleaning with Organic Solvents (i.e., BABB)

Perhaps the oldest and most common solvent clearing technique is called BABB (benzyl alcohol/benzyl benzoate). Here, tissues are cleared transparent by mixing benzyl alcohol and benzyl benzoate. The method is relatively easy and cheap, which is why it is a good option for cleaning tiny samples. It is especially useful

• The Dibenzyl Ether (DBE) Method

Another popular solvent-based technique is the dibenzyl ether (DBE) method. Dibenzyl ether is a less toxic alternative to BABB and offers excellent transparency without significant tissue shrinkage. It is commonly used for clearing nervous tissues, including brain slices, and is known for maintaining the integrity of both protein and nucleic acid markers.

DBE is the main issue, because it takes a couple of days to clear and it's longer than other options. And DBE can only penetrate hard tissue, making it ineffective at removing larger or more intricate organs.

Water-Based Tissue Clearing

Tissue clearing using water is usually safer than solvent based cleaning, and can be used to remove tissues while still maintaining their biology. These generally involve reducing scattering by changing tissues' refractive index to that of water without the need for harsh solvents.

• CLEARITY (Clear Lipid-exchanged Anatomic Resealed Tissue)

Perhaps the biggest innovation in tissue clearing was the CLARITY technique. CLARITY's gel-like hydrogel matrix is injected into tissue where the molecular structure remains intact, but the tissue becomes transparent in the optical sense. But CLARITY also cleans the lipids, proteins, and nucleic acids that are vital for biology.

This technique has become common in neuroimaging, where it can now see the brain at the microscopic level – even in complex and large samples. But it's slow, and needs careful experimental management for best results.

• ClearT (Clear Tissue)

In the ClearT method, a refractive index-matching solution made up of water, glycerol and other chemicals is applied. It's mainly used to dislodge relatively small specimens, like mouse tissue or plants. The ClearT procedure is both simple and quick with tissue clearing usually happening in a matter of hours.

However, it is limited by its applicability to small tissues and its relatively low penetration depth. While ClearT is effective for fluorescence-based imaging, it may not be suitable for large or dense tissues, where deeper penetration is needed.

• Scale (Scale A, B, and U)

Scale method (and scale variants Scale A, Scale B, and Scale U) – Scale is a common water-based tissue-clearing technique that uses a series of reagents to slowly cleave tissue. This is especially useful for sample size — whole organs, whole embryos.Scale uses chemicals like urea and guanidine, which reduce the tissue's refractive index mismatch, making it transparent.

Scale's flexibility, high tissue compatibility, and ability to maintain the integrity of molecular markers have made it one of the go-to methods for clearing organs in developmental biology and neuroscience research. However, the technique can lead to some loss of structural detail in particularly complex tissues, which limits its applicability in certain imaging scenarios.

Lipophilic and Chemical-Based Tissue Clearing

Lipophilic tissue clearing techniques aim to use lipid-based or chemical-based approaches to clear tissues. These methods are often designed to improve tissue penetration, increase transparency, and preserve tissue structure.

• PACT (Passive Clarity Technique)

The PACT method is another breakthrough in tissue clearing that focuses on passive lipid extraction. PACT removes lipids from tissue samples without the need for harsh chemical treatments. This technique involves using a hydrophilic reagent that extracts lipids passively, which allows the tissue to maintain structural integrity and enables better molecular marker retention.

PACT is often used in combination with other clearing methods, such as CLARITY or Scale, to improve the transparency of tissues while preserving fluorescent signals. While the PACT method is effective, it typically requires specialized equipment and reagents that are not always readily available in all laboratories.

• TDE (Tetrahydrofuran-Dimethyl Sulfoxide Clearing)

In clearing techniques using TDE, ethoxyfluoroethylene (THF) and dimethyl sulfoxide (DMSO) are the principal solvents that dissolve the lipids and proteins in the tissue to make it transparent. It's especially useful for severing stiffer tissues, like bones, and is common for skeletal and vascular imaging.

Despite its effectiveness in clearing dense tissues, TDE has its limitations, including the potential for toxicity and its less effective preservation of protein and nucleic acid integrity compared to other methods.

Hybrid and Multi-Step Tissue Clearing Techniques

In some cases, combining multiple clearing approaches yields superior results, especially for larger tissues or specimens that are challenging to clear with a single method. Hybrid methods combine solvent-based and water-based techniques, leveraging the strengths of each.

• TissueMAP (Tissue Matrix-Assisted Protocol)

The TissueMAP technique represents an emerging hybrid method where the tissue is initially subjected to a solvent-based clearing method to remove large lipids, followed by a water-based refractive index-matching solution for fine-tuning transparency. This hybrid approach is particularly useful for clearing tissues with complex structures, such as tumors, where both transparency and molecular preservation are critical.

While hybrid techniques like TissueMAP provide superior results in specific scenarios, they can be more labor-intensive and require precise optimization to balance the clearing effects and tissue integrity.

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Conclusion

It all depends on the tissue sample size, resolution, imaging technique (flavourescence or electron microscopy), and molecular markers being examined, to decide on which tissue clearing method.

Water-based solutions such as BABB and DBE can be used for smaller tissues; whereas water-based solutions like CLARITY and Scale can be used for larger samples. Lipophilic and chemical-based techniques, such as PACT or TDE, are better for applications with a more specialized use case, like skeletal tissue image or organ transparency. Hybrid techniques take the best of each technique and are more adaptable for more difficult biology.

Ultimately, the explosion of tissue clearing techniques has allowed tissues and organs to be visualized at ever higher resolution, and it can now be used to learn about developmental biology, disease mechanisms and therapeutics. The more sophisticated the tissue clearing techniques, the more powerful the instruments with which to study the complexities of living life.

1. Zhan YJ, et al.; Tissue Clearing and Its Application in the Musculoskeletal System. ACS Omega. 2023, 8(2):1739-1758.

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