Introduction

The field of biological and medical imaging has always led the way – it provides unparalleled information about the intricate internal and external anatomy of tissues and organs. Yet one of the remaining bioimaging obstacles is biological tissue, a veil that keeps the deep structure of cells and subcellular entities hidden from view. Even classical techniques such as sectioning and staining aren't quite enough to capture the whole three-dimensional (3D) image. Enter hydrogel-based tissue clearing, a process that's changed the face of bioimaging by making tissues translucent but structural and molecular properties intact. In this article, we discuss the theory, applications, and future of tissue clearing with hydrogels in bioimaging.

Figure 1. Tissue clearing using hydrogel-based CLARITY method. (Frenkel N, et al.; 2023)

The Need for Tissue Clearing

Biological tissues are by nature transparent because they are made of several light-scattering substances such as proteins, fats and water. The opacity of the substance also inhibits the penetration depth of light, which is why imaging of thick materials is very difficult. Traditional histology is the process of separating tissues into fine fragments, then stained and under the microscope. While good for some purposes, this method takes a long time, it is time-consuming, and often spatial context is lost.

These are all overcome by tissue clearing that leaves tissues clear so that we can deep-image without sectioning. This not only preserves the sample's 3D structure, but also allows high-resolution imaging techniques such as confocal microscopy, light-sheet fluorescence and multiphoton microscopy.

Principles of Hydrogel-Based Tissue Clearing

Hydrogel-based tissue clearing: Embedding living tissues in hydrogel, to stabilize tissue and avoid tissue shrinkage or disfiguration, allows clearing of tissues. Hydrogel embedding, removal of lipids, and refractive index match are the most important parts of this method.

1. Hydrogel Embedding

The first is to inject the tissue with a hydrogel precursor solution, which is usually made of acrylamide and bis-acrylamide. The solution goes through the tissue, polymerization is triggered and the hydrogel network continues to stick to the sample. This hydrogel structure holds the tissue in place mechanically and shields the tissue from structural damage in the next steps.

2. Lipid Removal

It is by far the most important light scatterer in biological tissues: lipids. : These lipids have to be removed to be transparent. That's usually done with detergents such as sodium dodecyl sulphate (SDS) or organic solvents that dissolve and extract lipids from the tissue without affecting other macromolecules such as proteins and nucleic acids.

3. Refractive Index Matching

Even when the lipids have been removed, refractive index difference between tissue elements can scatter light. This is reduced by immersing the clear tissue in a refractive index matching solution, which makes all the tissue parts optically equivalent. We usually find refractive index matching solutions like glycerol, sucrose and iohexol.

Advantages of Hydrogel-Based Tissue Clearing

The benefit of tissue clearing with hydrogels compared to other types of clearing:

• Structural Integrity Maintenance: The hydrogel structure keeps the tissue from shrinking or deforming which is a key to 3D imaging.
• Working with Many Tissues: You can use this technique on any tissue from biopsies to organs and can use it for any research application.
• Superior Transparency: Hydraulic clearing is particularly transparent to allow for deep tissue imaging because it successfully removes lipids while balancing refractive indexes.
• Preservation of Molecular Data: While some clearing techniques damage or alter biomolecules, hydrogel clearing stores proteins, nucleic acids, and other important molecules for molecular analysis later.

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Applications in Bioimaging

Tissue cleansing via hydrogel has presented a whole new avenue of biological and medical investigation. Some key applications include:

1. Neuroscience

Brains are so 3D, so cellularly packed that it is an obvious target for tissue destruction. Clearing by hydrogels – including CLARITY (Clear Lipid-exchanged Acrylamide-hybridised Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue hYdrogel) – was used to map neural networks, brain connectivity and research neurodegenerative disorders. They can now photograph whole mouse brains, and trace the wiring within neurons and see how regions of the brain interact with each other.

2. Developmental Biology

A picture of how organisms develop needs 3D images of tissues and organs at every stage. Using hydrogel-based tissue clearing, one can observe development in whole embryos or whole sections of large tissue – organogenesis, cell movement and tissue formation can all be observed.

3. Cancer Research

Tissue from tumours is very heterogeneous and possesses many different microenvironments that control the progression and spread of cancer. Through hydrogel clearing, whole tumours and stroma can be imaged to investigate tumour structure, invading fronts, and the relationships between cancer cells and the immune system.

4. Cardiovascular Research

The heart and vascular system are not simple to 3D-model as they are so complex. Tissue clearing using hydrogel can be used to image entire hearts and large blood vessels, which can be used to study cardiovascular development, mechanisms of disease and therapeutic effect.

Challenges and Future Directions

For all its benefits, tissue clearing with hydrogel is not perfect. These are some of the main ones:

• Time: This can be tedious especially if it's larger tissues or organs. Teams are working on faster clearing without sacrificing transparency or structure.
• Standardization: Difference in clearing protocol and reagent can result in non-standard results. Standardised protocols and reagents would make it more reproducible and compareable study to study.
• Imaging Techniques Supported: Hydrogel clearing can be applied to many imaging techniques but optimizing for certain imaging techniques, and for image resolution and contrast enhancement, are still areas of study.
• Molecular Labeling: Keeping molecular labels, like fluorescent antibodies or genetic reporters, intact and visible throughout clearing is essential to image accurately.

In the future, hydrogel clearing in combination with other technologies, including high-resolution microscopy and computational simulations, will add to our knowledge about biology. Combining removed tissues with light-sheet microscopy, for example, can rapidly image large volumes, and machine learning algorithms can also help compliancy complex datasets from 3D images.

Conclusion

Hydrogel tissue clearing revolutionised bioimaging because it has given us a tool to look at 3D structure of living tissues in a way never before seen. From the map of brain circuits to the pathogenesis of cancers and development, it's a technique with many applications that are revolutionising our knowledge about complex biological systems. Although there are still challenges, continuous advancements and the adoption of complementarity will only make hydrogel tissue clearing further powerful in the future. This open revolution in bioimaging will only continue to illuminate the secret worlds of life and death.

1. Frenkel N, et al.; Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver. Front Oncol. 2023, 13:1062926.

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