Tissue Extraction in Bioimaging via Solvent Based Tissue Clearing

The one problem with bioimaging is that the tissues are opaque, which makes imaging only half-asthentic. Tissue clearing methods have evolved to counter this, of which solvent tissue clearing is one. In this paper, I review the concepts, techniques, and applications of solvent tissue clearing in bioimaging and its potentials and drawbacks.

Principles of Tissue Clearing

Tissue clearing is a process of making biological tissue transparent for deep imaging, but retaining the structure and molecular content of the samples. The main barrier to transparency is the variation in refractive index between cell structures and the medium they exist in. Solvent clearing solves this by cleaning the surfaces with organic solvents, which make the refractive indices homogenous, and so suppress light scattering and absorption.

Solvent-Based Clearing Techniques

Figure 1. Organic/hydrophilic solvent-based methods. (Lee E, et al.; 2016)

1. BABB (Benzyl Alcohol and Benzyl Benzoate)

BABB is one of the earliest solvent-based clearing procedures: a combination of benzyl alcohol and benzyl benzoate. Tissues are dried in a graduated series of alcohols, then submerged in BABB, which renders the tissue transparent. This technique is straightforward and efficient but will shrink tissue massively and bleed fluorescence.

2. 3DISCO (Three-Dimensional Imaging of Solvent-Cleared Organs)

3DISCO is even better than BABB because there are more steps to maintain fluorescence. Tissues are sucked dry with tetrahydrofuran (THF) and then rinsed with dibenzyl ether (DBE). This technique is transparent and fluorescence-preserving, which makes high resolution imaging possible. But it will still shrink tissue and is incompatible with storage over time.

3. iDISCO (Immunolabeling-enabled 3DISCO)

iDISCO improves upon the 3DISCO method with immunolabeling before clearing. This technique allows for specialized labelling of proteins and other molecules in the tissue, for highly specific structural and functional studies. iDISCO is already commonly used in neuroscience and developmental biology, but it needs to be calibrated with antibody levels and clearing conditions.

4. uDISCO (Ultimate DISCO)

uDISCO promotes tissue clearance with a series of organic solvents that result in very low tissue shrinkage and high fluorescence retention. Tissues dehydrate in THF and clear in BABB and DBE. uDISCO is ideal for large tissue slices and whole-organ scanning but involves toxic chemicals that need to be handled with care.

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Applications in Bioimaging

1. Neuroscience

As a method of brain mapping, tissue clearing using solvents is used in neuroscience. Such techniques as iDISCO and uDISCO enable high-resolution imaging of whole brains, where researchers can study neuronal organisation and connectivity at the smallest level possible. These technologies allow fluorescently labelled neurons, synapses and proteins to be visualized to learn about the brain and its disorders.

2. Developmental Biology

Growth biology is also helped by solvent-based clearing – in which you can see developing embryos and tissues. When immunolabeling is combined with clearing approaches such as iDISCO, it's possible to map the location and time of gene expression, protein distribution and cellular interactions over development. This helps explain how embryogenesis, organogenesis and tissue differentiation happen.

3. Cancer Research

Solvent-based tissue clearing is equally useful in cancer studies, where it is used to see the tumour structure, vascular structures and metastatic networks. Clearing methods make it possible to study whole tumors and their microenvironments, learning more about the way tumours grow, invade and respond to treatment. This can lead to better cancer diagnosis, treatment and biology of the tumour.

4. Organ and Tissue Engineering

Solvent clearing methods in organ and tissue engineering allow engineers to see engineered tissues and organs. These methods can measure cell organisation, vascularisation and integration with host tissues. Solvent-based clearing allows high-resolution imaging of complex, large samples allowing tissue-engineered structures to be developed and optimized for regenerative medicine.

Advantages and Limitations

Advantages

1. Clearing with Solvent: During clearance, you can observe deep tissues, large samples and organs.
2. High Resolution: These methods retain small structural granularity and allow high resolution imaging of cellular and subcellular structures.
3. Fluorescence-Compatibility: Techniques such as 3DISCO and iDISCO do not remove fluorescence and can be used for fluorescent markers and labels.
4. Propagation: Solvent-based clearing can be used on many different tissues and biological material and is an omnipresent tool in bioimaging.

Limitations

1. Tissue Shrinkage: A lot of solvent clearing procedures shrink a lot of tissues, leading to anatomical distortion.
2. Chemical Toxicity: Organic solvents that are used in these techniques are often toxic and must be handled and disposed of appropriately.
3. Fluorescence Quenching: Fluorescence can be quenched with long exposure to cleaning solvents which inhibits imaging times.
4. Optimization: All clearing protocols must be optimised, from dehydration and solvent quantities to incubation times.

Future Perspectives

Solvent tissue clearing is a science in progress, and researchers are constantly finding new ways to make them transparent, less toxic and fluorescence-preserving. Tissue clearing is likely to be enhanced by solvent design, imaging and more. Additionally, clearing together with other imaging techniques like light-sheet microscopy and optical coherence tomography could help widen the scope of solvent-based clearing for biomedical studies.

Conclusion

Solvent-driven tissue clearing is a great bioimaging tool, and helps us view highly detailed, high resolution biological structures. Although it is flawed, the method has a wide range of applications in neuroscience, developmental biology, cancer research and tissue engineering. With more researchers enhancing and perfecting these techniques, solvent-based tissue clearing will be even more important to our knowledge of biology and to biomedical science.

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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