CLARITY Brain: Tissue Clearing for Detailed Connectivity

Studying the brain’s intricate network requires methods that allow researchers to see deep into tissue while preserving structural and molecular details. Traditional techniques involve slicing thin sections, which can disrupt spatial relationships between neurons. To overcome this, scientists have developed tissue-clearing methods that render whole brains or large samples transparent while maintaining their architecture.

CLARITY is one such technique, enabling high-resolution imaging of neural circuits without physical sectioning. By making tissues optically clear while retaining proteins and nucleic acids for labeling, CLARITY has revolutionized neuroscience research.

Key Steps In Hydrogel Embedding

Embedding brain tissue in a hydrogel matrix stabilizes cellular structures while allowing lipid removal, which hinders optical transparency. This process begins with infusing a hydrogel monomer solution—typically acrylamide, bisacrylamide, and formaldehyde—into the tissue. Formaldehyde crosslinks proteins and nucleic acids to the hydrogel, ensuring molecular components remain anchored during clearing. The acrylamide concentration is optimized to balance rigidity and permeability, as excessive polymerization can impede clearing agents.

Once infused, polymerization is initiated using thermal or chemical catalysis, often with ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). Passive polymerization at lower temperatures minimizes protein denaturation, preserving antigenicity for immunostaining. The polymerized hydrogel immobilizes biomolecules while leaving lipid membranes unbound, preparing them for extraction.

Preventing oxygen inhibition is critical, as it can interfere with polymerization. Tissues are incubated in a low-oxygen environment, such as a vacuum-sealed chamber. Hydrogel composition can also be adjusted for different tissue types, with softer hydrogels preferred for delicate structures like the hippocampus and denser formulations providing stability for larger specimens.

Detergent Based Lipid Removal

Once the hydrogel matrix is established, lipids must be removed to enhance optical transparency. Lipids scatter light, impeding deep tissue imaging. Removing them requires detergents that solubilize membranes while preserving proteins and nucleic acids within the hydrogel.

Sodium dodecyl sulfate (SDS) is the most commonly used detergent. As an anionic surfactant, SDS integrates into lipid membranes, leading to solubilization. The efficiency of lipid removal depends on SDS concentration, temperature, and incubation duration. Typically, a 4–8% SDS solution is used, with higher concentrations accelerating extraction but risking protein denaturation. Temperature is maintained between 37°C and 50°C to balance lipid clearance with molecular preservation.

Passive clearing involves incubating tissue in SDS for extended periods, allowing gradual diffusion. While minimizing shear stress, this requires frequent buffer exchanges. Active clearing, such as electrophoretic tissue clearing (ETC), accelerates lipid removal by applying an electric field to drive SDS migration. ETC significantly reduces clearing time but requires precise voltage regulation—typically 20–50 V—to prevent hydrogel degradation or protein loss.

Optical Clearing And Refractive Index Matching

Achieving optical transparency requires more than lipid removal; it also involves addressing light scattering from mismatched refractive indices. Biological tissues contain proteins, nucleic acids, and extracellular matrix structures, each with distinct refractive properties. When light encounters these variations, it bends unpredictably, distorting images and reducing penetration depth. Refractive index matching solutions (RIMS) homogenize optical properties, allowing light to travel with minimal scattering.

The choice of clearing medium is crucial for transparency and molecular integrity. Aqueous-based RIMS, such as FocusClear and ScaleS, are commonly used due to their compatibility with fluorescent labeling. These solutions adjust the refractive index to approximately 1.45, closely matching that of fixed tissue. High-index organic solvents like BABB (benzyl alcohol and benzyl benzoate) and DBE (dibenzyl ether) offer superior transparency but can cause protein denaturation or fluorescence quenching, making them less suitable for long-term imaging.

Immersion time and solution exchange frequency influence clearing effectiveness. Tissue samples are incubated in RIMS for hours to days, depending on size and density. Advances in clearing protocols have led to hybrid approaches, such as SeeDB and FRUIT, which combine the transparency benefits of organic solvents with the molecular preservation advantages of aqueous solutions. These methods enable large-volume imaging of intact brains without sectioning.

Visualization Of Neuronal Connections

Mapping neuronal connections with high precision requires imaging techniques that capture intricate axonal and dendritic projections while preserving spatial relationships. Advances in fluorescence microscopy, particularly light-sheet and confocal approaches, enable detailed visualization of neural circuits. Light-sheet microscopy selectively illuminates thin sections of cleared tissue, minimizing photobleaching and enhancing signal-to-noise ratios, making it well-suited for large-volume imaging. Confocal and two-photon microscopy offer higher resolution but require longer acquisition times due to point-by-point scanning. Adaptive optics further improve image clarity by correcting aberrations from residual refractive index mismatches.

Fluorescent labeling strategies distinguish individual neurons and their projections. Genetically encoded markers like Brainbow use combinatorial fluorescent protein expression to assign unique colors to neighboring neurons, aiding circuit tracing. Immunolabeling with antibodies targeting synaptic proteins, such as synaptophysin or PSD-95, identifies connectivity patterns, while viral tracers like rabies and AAV-based constructs enable transsynaptic labeling. Combining these approaches with expansion microscopy enhances resolution, allowing subcellular structures to be visualized at near-electron microscopy levels without ultrathin sectioning.

Preservation Of Molecular Signals

Ensuring molecular markers remain intact throughout the CLARITY process is essential for downstream analyses, including immunostaining, fluorescent in situ hybridization (FISH), and transcriptomic profiling. Lipid removal and refractive index matching can disrupt protein epitopes and nucleic acid structures, requiring optimized conditions for preservation. Formaldehyde crosslinking during hydrogel embedding stabilizes proteins and nucleic acids but can mask epitopes, reducing antibody accessibility. Antigen retrieval techniques, such as heat-induced epitope recovery or enzymatic digestion, restore binding sites for immunolabeling. Buffer pH and ionic composition are adjusted to maintain RNA integrity, particularly for transcript detection.

Fluorescence retention is another challenge, as prolonged exposure to detergents and high refractive index solutions can cause quenching or photobleaching. To counteract this, CLARITY protocols incorporate stabilizers like trolox or ascorbic acid to minimize oxidative damage. Alternative labeling strategies, such as enzymatic signal amplification or quantum dots, enhance fluorescence durability for long-term imaging. Hybrid clearing protocols combining CLARITY with methods like iDISCO or SHIELD improve molecular preservation while maintaining transparency. These refinements expand CLARITY’s applications, enabling researchers to map neuronal architecture and investigate gene expression, protein localization, and disease-associated molecular changes in intact brain tissue.