As scientists continue to study disease states in a variety of tissues, an understanding of the 3D structure of organs is providing insight into structure-function relationships that affect tissue functioning. Advanced imaging techniques are giving researchers a better view into the inner workings of complex tissues, but some significant obstacles still stand in the way of truly comprehensive tissue imaging. One such obstacle, the inherent opacity of tissue, has proved particularly difficult to overcome. Ultimately, if light can’t penetrate a tissue, there is no way to image it.
Options for tissues imaging
One solution to this challenge has been sectioning tissue into slices thin enough to be imaged using traditional microscopy techniques, and then using sequential images to reconstruct a virtual 3D image. This is a useful tool, but it has drawbacks. Tissues can be irreparably folded or torn during sectioning, and this disruption affects the reconstructed 3D image. In addition, producing intact sections is a difficult process that requires time and, in some cases, a highly trained scientist. Thus it is an inefficient process.
Blockface methods also have been used with some success: The surface of a block of tissue is imaged and then shaved off to reveal the next layer of tissue. This process has the advantage of not disrupting the tissue prior to imaging, but each layer of tissue is destroyed as soon as it is imaged, limiting the usefulness of this technique.
The ultimate solution would be volume imaging without sectioning or otherwise disrupting the tissue, and scientists have searched for a methodology that would render tissue translucent while maintaining its structural integrity. With the invention of tissue clearing, and notably the CLARITY technique, it is now possible to achieve true volume imaging of intact tissue.
In addition to CLARITY, other tissue-clearing methods are available. Some of these methods, such as iDISCO and PACT, are compatible with small-molecule labeling of cleared tissues. Other methods, including CUBIC, Scale and SeeDB, do not allow for small-molecule labeling after the tissue is cleared, making experiments like immunostaining and FISH impossible to perform. Of the tissue-clearing methods available, CLARITY has the shortest experimental times, especially when it is run in a commercial system. This makes CLARITY a strong choice for researchers who need quick and reliable results as well as the ability to label samples after tissue clearing.
By virtue of the purpose they serve, lipid bilayers create barriers that make it difficult to image deep within intact tissue, specifically keeping the tissue opaque and milky and causing light scattering. They also create diffusion barriers that prevent small molecules from moving freely within tissue samples, which in turn poses a challenge for techniques such as immunostaining. But lipid bilayers are such an integral part of tissue that removing them while keeping the tissue intact for imaging has not been possible until the creation of CLARITY, which works by very selectively removing lipid bilayers from tissue.
How CLARITY works
CLARITY transforms intact tissue into a hydrogel-tissue hybrid construct that is perfectly intact and mechanically stable as well as permeable to small molecules and photons. Hydrogel crosslinks secure biomolecules, making it possible to remove lipids from the tissue without disrupting its structure.
CLARITY starts with a hydrogel monomer infusion into the tissue of interest. The tissue sample is incubated in a hydrogel solution to enable complete diffusion of the hydrogel into the tissue. After the tissue sample is fully infused with hydrogel, the hydrogel is polymerized, which crosslinks biomolecules into their location to completely preserve the molecular structure of the tissue.
After polymerization is complete, lipids are removed from the tissue to clarify it. This is performed either passively or actively, by placing samples in a detergent solution. Passive removal of lipids is less costly, but it is not a reliable method for larger tissue samples, such as entire organs. Active removal of lipids from tissue samples is faster and more efficient.
Active lipid removal occurs via electrophoresis. Samples are placed in a buffer containing SDS, and a current is applied across the sample while the buffer is continuously circulated. The exact time required for electrophoretic removal of lipids depends on a variety of factors, including the current applied and the temperature of the solution, but it can range from several hours to several days.
After clearing, tissues lose their milky white appearance and are ready for volumetric imaging. In addition to being optically transparent, cleared tissues are macromolecule permeable. Small molecules and biomolecules, including antibodies and RNA, can permeate the entire tissue.
CLARITY originally was developed to study the brain, and it continues to be an invaluable tool for neuroscience researchers studying deep sections of intact brains. More recently, CLARITY has been optimized for other organs [1].
Although CLARITY gives researchers unprecedented access to tissue imaging, there are some common difficulties scientists may face with electrophoretic tissue clearing. Poorly constructed clearing chambers with inadequate buffer circulation can leave bubbles trapped in the chambers, causing poor temperature control and inadequate clearing. When the tissue sample is overheated because of poor temperature control, irregular electrical currents or poor buffer circulation, damage to the tissue can include burning, melting and the formation of black precipitates. The conditions that cause these problems can be very difficult to correct manually.
Commercial solutions
To address these challenges, commercial solutions for performing CLARITY are available. These systems provide an all-in-one solution that takes the guesswork and protocol development out of the process and enables researchers to clear tissue for imaging rapidly and consistently. The use of a commercial system also reduces the amount of time CLARITY requires, from up to two weeks for a mouse brain to just eight hours.
Studying the structural and functional relationships of complex tissues remains an important field of research. With the application of CLARITY, scientists can gain unprecedented insight into tissues and disease states.
Reference
[1] Lee, H, et al., “Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung and intestine,” BMC Developmental Biology, 14, 2014.[PMID: 25528649]
Image: X-CLARITY™ ETC Chamber and Controller, Logos Biosystems
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