# Tissue clearing

Tissue clearing refers to a group of chemical techniques used to turn tissues transparent. By turning tissues transparent to certain wavelengths of light, it allows one to gain optical access to a tissue. That is, light can pass into and out of the cleared tissue freely, allowing one to see the structures deep within the tissue without physically cutting it open. Many tissue clearing methods exist, each with different strengths and weaknesses. Some are generally applicable, while others are designed for specific applications. Tissue clearing is usually useful only combined with one or more fluorescent labeling techniques such as immunolabeling and subsequently imaged, most often by optical sectioning microscopy techniques. Tissue clearing has been applied to many areas in biological research. It is one of the more efficient ways to perform three-dimensional histology.

## History

In the early 1900s, Werner Spalteholz developed a technique that allowed the clarification of large tissues, using Wintergrünöl (methyl salicylate) and benzyl benzoate. Various scientists then introduced their own variations on Spalteholz's technique. Tuchin et al. introduced tissue optical clearing (TOC) in 1997, adding a new branch of tissue clearing that was hydrophilic instead of hydrophobic like Spalteholz's technique. In the 1980s, Andrew Murray & Marc Kirschner developed a two-step process, wherein tissues were first dehydrated with alcohol and subsequently made transparent by immersion in a mixture of benzyl alcohol and benzyl benzoate (BABB), a technique they coupled with light sheet fluorescence microscopy, which remains the method with the highest clearing efficacy to date, regardless any tissue pre-processing step. In the most extreme case, it allows the clearing of a whole mouse of even a whole human brain.

## Principles

Tissue opacity is thought to be the result of light scattering due to heterogeneous refractive indices. Tissue clearing methods chemically homogenize refractive indices, resulting in almost completely transparent tissue.

## Classifications

While there are multiple class names for tissue-clearing methods, they are all classified based on the final state of the tissue by the end of the clearing method. These include hydrophobic clearing methods, which may also be known as organic, solvent-based, organic solvent-based, or dehydration clearing methods; hydrophilic clearing methods, which may also be known as aqueous-based or water-based methods, and hydrogel-based clearing methods.

## Labeling

Tissue clearing methods have varying compatibility with different methods of fluorescent labeling. Some are better suited to genetic labelling by endogenously expressed fluorescent protein, while others externally delivered probes as immunolabeling and chemical dye labeling. The latter is more general and applicable to all tissues, notably human tissues, but the penetration of the probes becomes a critical problem.

## Imaging

After clearing and labeling, tissues are typically imaged using confocal microscopy, two-photon microscopy, or one of the many variants of light-sheet fluorescence microscopy. Other less commonly used methods include optical projection tomography and stimulated Raman scattering. As long as the tissue allows for the unobstructed passing of light, the optical resolution is fundamentally limited by Abbe diffraction limit. The compatibility of any tissue clearing method with any microscopy system is, therefore, configurational rather than optical.

## Data

Tissue clearing is one of the more efficient ways to facilitate 3D imaging of tissues, and hence generates massive volumes of complex data, which requires powerful computational hardware and software to store, process, analyze, and visualize. A single mouse brain can generate terabytes of data. Both commercial and open-source software exists to address this need, some of it adapted from solutions for two-dimensional images and some of it designed specifically for the three-dimensional images produced by imaging of cleared tissues.

## Applications

Tissue clearing has been applied to the nervous system, bones (including teeth), skeletal muscles, hearts and vasculature, gastrointestinal organs, urogenital organs, skin, lymph nodes, mammary glands, lungs, eyes, tumors, and adipose tissues. Whole-body clearing is less common, but has been done in smaller animals, including rodents. Tissue clearing has also been applied to human cancer tissues. For some techniques, bone tissue must be decalcified to remove light-scattering hydroxyapatite crystals, leaving behind a protein matrix suitable for clearing.