- CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel) is a groundbreaking tissue clearing method that transforms intact biological tissues into optically transparent and permeable structures while preserving their molecular and structural integrity. Developed by Karl Deisseroth and colleagues at Stanford University, this technique has revolutionized our ability to study complex biological structures in three dimensions.
- The fundamental principle of CLARITY involves embedding tissue in a hydrogel matrix that preserves its structural and molecular components. The process begins by infusing the tissue with a mixture of acrylamide monomers, bisacrylamide, and formaldehyde. When heated, these compounds polymerize to form a hydrogel mesh that crosslinks with biomolecules like proteins and nucleic acids, while leaving lipids unbound. This selective binding is crucial for maintaining the tissue’s structural integrity while allowing lipid removal.
- The next critical step involves removing lipids through electrophoretic tissue clearing (ETC) or passive thermal clearing. In ETC, an electric field drives charged detergent micelles through the tissue, actively removing lipids while leaving the hydrogel-bound components intact. Passive clearing achieves the same result through simple diffusion at elevated temperatures, though it takes longer. The removal of lipids dramatically reduces light scattering, rendering the tissue transparent.
- A key advantage of CLARITY is its ability to preserve proteins, nucleic acids, and other biomolecules in their native locations while making the tissue accessible to molecular probes. The hydrogel mesh maintains spatial relationships between molecules while creating a permeable structure that allows antibodies and other labels to penetrate deeply into the tissue. This enables multiple rounds of staining and imaging of the same specimen.
- The technique has found particular application in neuroscience, where it allows researchers to trace neural connections and study protein distributions throughout entire brains. The ability to image intact neural circuits in three dimensions has provided new insights into brain organization and function. CLARITY has revealed previously unknown features of brain connectivity and helped researchers understand how neural circuits are altered in various diseases.
- Sample preparation is critical for successful CLARITY implementation. The tissue must be properly fixed and uniformly infused with the hydrogel solution. Temperature control during polymerization is crucial, as is the careful removal of oxygen, which can inhibit the polymerization process. The clearing process must be monitored to prevent tissue damage while ensuring complete lipid removal.
- Recent modifications to the original CLARITY protocol have improved its utility and accessibility. PACT (Passive CLARITY Technique) eliminates the need for specialized electrical equipment by relying on passive clearing. PARS (Perfusion-assisted Agent Release in Situ) adapts the technique for whole-body clearing through the circulatory system. These variations have made the technique more versatile and easier to implement.
- Imaging cleared tissues requires specialized microscopy techniques. Light-sheet fluorescence microscopy is particularly well-suited for CLARITY samples, offering rapid acquisition of large tissue volumes with minimal photobleaching. Advanced objective lenses with long working distances and correction for refractive index matching solutions are typically required for optimal imaging.
- Data analysis presents significant challenges due to the large size of three-dimensional imaging datasets. Specialized software tools have been developed to handle the terabyte-scale data generated from CLARITY imaging. These tools enable three-dimensional reconstruction, automated feature detection, and quantitative analysis of cellular and molecular distributions.
- The technique has expanded beyond neuroscience into other fields of biological research. CLARITY has been adapted for studying development, cancer biology, and various organ systems. The ability to maintain molecular information while achieving tissue transparency has made it valuable for understanding complex biological processes in their native context.
- Applications in clinical research are growing, with adaptations of CLARITY being used to study human tissue samples. This has provided new insights into disease processes and potential therapeutic targets. The technique’s ability to reveal three-dimensional tissue organization while maintaining molecular information makes it particularly valuable for understanding pathological changes.
