CLARITY

Several published papers have applied the CLARITY method to a wide range of tissues and disease states such as immuno-oncology for human breast cancer, Alzheimer's disease human brains, mouse spinal cords, multiple sclerosis animal models, and plants. CLARITY has also been combined with other technologies to develop new microscopy methods including confocal expansion microscopy, SPIM light sheet microscopy, and CLARITY-optimized light sheet microscopy (COLM).

Procedure

The process of applying CLARITY imaging begins with a postmortem tissue sample. Depending on the tissue type pre-processing steps such as demineralization or decolorization may be necessary after sample fixation. Next a series of chemical treatments must be applied to achieve transparency, in which the lipid content of the sample is removed, while almost all of the original proteins and nucleic acids are left in place. The purpose of this is to make the tissue transparent and thus amenable to detailed microscopic investigation of its constituent functional parts (which are predominantly proteins and nucleic acids). To accomplish this, the preexisting protein structure has to be placed in a transparent scaffolding which preserves it, while the lipid components are removed. This 'scaffolding' is made up of hydrogel monomers such as acrylamide. The addition of molecules like formaldehyde, paraformaldehyde, or glutaraldehyde can facilitate attachment of the scaffolding to the proteins and nucleic acids that are to be preserved, and the addition of heat is necessary to establish the actual linkages between the cellular components and the acrylamide.

Once this step is complete, the protein and nucleic acid components of the target tissue's cells are held firmly in place, while the lipid components remain detached. Lipids are then removed over days to weeks for passive diffusion in detergent, or accelerated by electrophoretic methods to only hours to days. Efforts to accelerate by electrophoretic methods remain inconclusive on ability to not cause tissue damage. As they pass through, the detergent's lipophilic properties enable it to pick up and excise any lipids encountered along the way. Lipophilic dyes as DiI are removed, however there are CLARITY-compatible lipophilic dyes that can be fixated to neighbouring proteins. The large majority of non-lipid molecules, such as proteins and DNA, remain unaffected by this procedure, thanks to the acrylamide gel and chemical properties of the molecules involved.

As reported in the initial paper, the tissue expands during this process, but as needed can be restored to its initial dimensions with a final step of incubation in refractive index matching solution. Tissue expansion does occur in the uniquely lipid-rich brain; however, other tissue types have been noted to not experience as much tissue expansion throughout the process.

By this stage in the process, the sample has been fully prepared for imaging. The contrast for imaging can come from endogenous fluorescent molecules, from nucleic acid (DNA or RNA) labels, or from immunostaining, whereby antibodies that bind specifically to a certain target substance are used. In addition, these antibodies are labeled with Fluorescent tags that are the key to final imaging result. Standard confocal, two-photon, or light-sheet imaging methods are all suitable to then detect the fluorescence emitted down to the scale of protein localization, thus resulting in the final highly detailed and three-dimensional images that CLARITY produces.

After a sample has been immunostained for an image, it is possible to remove the antibodies and re-apply new ones, thus enabling a sample to be imaged multiple times and targeting multiple protein types.