Fluid-based tissue clearing has been a game-changing tool in bioimaging that enables scientists to look at and study biological tissues with new levels of resolution and granularity. In it, the biological tissue becomes transparent but is structurally and molecularly intact so deep tissue imaging with little light scattering can be performed. Aqueous clearing methods aren't reliant on the use of toxic organic solvents, because they are based on water, and can be applied to a wider variety of biological samples and imaging techniques.

Introduction to Tissue Clearing

Tissue clearing is used to clear tissues, so that cell and subcellular structures can be viewed in three dimensions. The idea here is to minimize light scattering and absorption, which are the primary challenges when imaging the deep tissues. Classic histology is the cut-and-peel process in which tissue is cut up and sliced off, removing the original structure of the sample and restricting its analysis. Tissue clearing, by contrast, allows the investigation of whole organs or massive quantities of tissue with intact samples, which is much more encompassing of biology.

Figure 1. The tissue is placed in an aqueous clearing solution for days to months, and the solution is exchanged during the process.(Lee E, et al.; 2016)

Principles of Aqueous-Based Tissue Clearing

Tissue clearing works according to a theory that the RI of the tissue is matched to that of the medium. Tissues in the body are constituted by different molecules that have different RIs and so light scatters when it is illuminated. Because the RI is homogenised over the entire sample, the light doesn't reflect very much and it makes the tissue transparent.

Clearing procedures using water do so by enacting water-soluble compounds that work on tissues and change their optical properties. These are usually detergents, hydrophilic polymers, and other chemicals that react with tissue components to equalise the RI without massive damage or shrinkage.

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Advantages of Aqueous-Based Tissue Clearing

Tissue clearing involving water has several advantages over conventional organosolvent methods:

1. Biocompatibility: Water based solutions are less toxicity and less likely to result in the denaturation of proteins and other biomolecules preserving the structural and molecular integrity of the tissue.
2. Fluorescence Imaging: Most water-based clearing agents are compatible with fluorescent proteins and dyes and preserve the fluorescence signal, which permits for multi-color imaging.
3. Simple Installation: Aqueous encapsulation procedures are typically less complicated and less hazardous than organic solvent cleaning techniques as they typically don't involve many steps and/or require less specialized equipment.
4. Universality: These techniques can be used on many tissues including brain, heart, lung and even whole animal like zebrafish embryos and rodents.

Common Aqueous-Based Clearing Methods

There have been different aqueous tissue-clearing methods with different features and applications. Let's go over a few of the most popular methods.

• CLARITY

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue hYdrogel) is a new technique that replaces lipids in the tissue with a hydrogel matrix. — This is done by infiltrating the tissue with a hydrogel precursor solution and polymerising it to make a firm mesh. The lipids are then stripped by electrophoresis, and a transparent, structurally sound tissue remains. CLARITY is especially suited to the imaging of the brain where fine neural structures can be retained and deep antibody penetration for immunostaining.

• CUBIC

For CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational Analysis), amino alcohols and detergents are used to clear the tissues. It is suitable for removing huge tissues and whole organs such as brain, heart and liver. CUBIC protocols are relatively fast and simple so most labs can use them. Also, CUBIC is interoperable with many fluorescent proteins and dyes that allow for fine-grained multicolour imaging.

• Scale

Scale: A chemical technique of tissues removal using concentrated urea and other chemicals. It is particularly known to preserve the fluorescence of genetically encoded indicators, useful for neuroimaging studies. Scale has already been used to disenable whole mouse brains and other organs, and to obtain very detailed photos of neural circuits and cells.

Applications in Bioimaging

Water-based tissue clearing changed bioimaging by allowing scientists to study the details of biological systems at unprecedented resolution. Some key applications include:

• Neuroscience

In the neurosciences, tissue clearing made it possible to map circuits and research brain connectivity. Such tools as CLARITY and CUBIC allowed scientists to create images of whole-brain structures and diagram neuronal circuits in a high degree of detail. It has wrought new knowledge about how the brain works and how neurological diseases operate.

• Developmental Biology

Clearing techniques have been useful for the study of embryonic growth and organogenesis. If whole embryos are transparent, then they can watch development in their natural setting and see when certain genes and proteins are expressed.

• Cancer Research

Tissue clearing also helped cancer studies visualize tumor microenvironments and interactions between cancer cells and tissues. This can help in tracking the development of the tumour, its spread and whether treatment is effective.

Future Directions

Tissue clearing is a dynamic process, and there is always an attempt to refine existing techniques and create new ones. Future directions include:

1. Advanced Imaging Strategies: Integrating tissue clearing with high-resolution and deep tissue permeability imaging techniques like light-sheet microscopy and multiphoton microscopy.
2. New Clearing Agents: Creating new clearing agents with faster clearance times, better fluorescence retention and improved compatibility with more tissues.
3. Standardization and Accessibility: Developing standard protocols and increasing the accessibility of clearing reagents to researchers around the world in order to bring these methods more broadly to life.

Conclusion

Tissue clearing using water is now a potent bioimaging tool that lets scientists view and examine biological tissues in 3D in unprecedented detail. Transparent and structurally and molecularly intact tissues were revealed in these methods that revolutionized the analysis of biological systems and diseases. With the field developing at a faster pace, aqueous tissue clearing is set to be increasingly influential in biomedical science and bring new information and scientific innovation.

1. Lee E, et al.; See-Through Technology for Biological Tissue: 3-Dimensional Visualization of Macromolecules. Int Neurourol J. 2016, 20(Suppl 1): S15-22.

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