Comprehensive Tissue Clearing Protocol: Key Steps for Imaging

Advancements in imaging techniques have made it possible to visualize complex biological structures in unprecedented detail. However, the natural opacity of tissues limits light penetration and image clarity. Tissue clearing methods address this issue by rendering samples transparent while preserving structural integrity for high-resolution imaging.

Optimizing a tissue clearing protocol requires careful consideration of multiple factors, from sample preparation to imaging conditions.

Tissue Preparation And Fixation

Proper tissue preparation and fixation are fundamental to achieving high-quality imaging in cleared samples. The process begins with careful dissection to minimize mechanical damage and preserve native structures. Freshly excised tissue is prone to degradation, making prompt fixation necessary to stabilize cellular components and prevent autolysis. The choice of fixative plays a key role in maintaining tissue architecture while allowing effective clearing. Formaldehyde-based solutions, such as paraformaldehyde (PFA), are widely used due to their ability to crosslink proteins while maintaining compatibility with clearing protocols.

Fixation time and concentration must be optimized based on tissue type and size. Over-fixation can lead to excessive crosslinking, which hampers clearing reagent penetration, while under-fixation risks structural degradation. For delicate tissues like the brain, a lower concentration of PFA (e.g., 4%) with extended incubation at 4°C enhances preservation without excessive hardening. Perfusion fixation, where fixative is introduced through the vasculature, ensures uniform distribution and minimizes inconsistencies, making it ideal for whole-organ studies. In contrast, immersion fixation is more practical for smaller tissue sections but requires longer incubation for adequate penetration.

Thorough washing after fixation removes residual fixative that could interfere with clearing agents. Phosphate-buffered saline (PBS) is commonly used for this step, sometimes supplemented with detergents like Triton X-100 to enhance membrane permeability. Maintaining consistent buffer conditions is also important for clearing efficiency. Some protocols incorporate post-fixation treatments, such as methanol dehydration or lipid removal, to enhance transparency, but these steps must be carefully balanced to prevent excessive tissue shrinkage or loss of antigenicity.

Available Clearing Approaches

Tissue clearing techniques improve optical transparency while preserving structural integrity. The choice of method depends on factors such as tissue type, imaging depth, and downstream applications. The three primary categories of clearing techniques are solvent-based, hydrogel-based, and aqueous-based methods, each with unique advantages and limitations.

Solvent-Based

Solvent-based clearing methods rely on organic solvents to remove lipids and reduce light scattering, significantly increasing transparency. Techniques such as BABB (benzyl alcohol and benzyl benzoate) and 3DISCO (3D imaging of solvent-cleared organs) are highly effective for large, lipid-rich tissues. The process typically involves dehydration with graded methanol or ethanol, followed by delipidation using solvents like dichloromethane. A final refractive index-matching step enhances optical clarity.

While solvent-based methods provide rapid and deep tissue clearing, they can cause shrinkage and protein denaturation, affecting fluorescence signal retention. To mitigate these effects, protocols such as iDISCO+ incorporate hydrogel stabilization or antibody labeling before clearing. These methods are particularly useful for whole-organ imaging in neuroscience and developmental biology. However, the use of organic solvents requires careful handling due to their toxicity and potential for fluorescence quenching.

Hydrogel-Based

Hydrogel-based clearing techniques, such as CLARITY and PACT (passive clarity technique), embed tissues in a hydrogel matrix before lipid removal. This approach preserves protein content, making it well-suited for immunostaining and molecular analysis. The process begins with fixation in a formaldehyde and acrylamide solution, followed by polymerization to create a hydrogel scaffold. Lipids are then extracted using detergents like sodium dodecyl sulfate (SDS), leaving behind a transparent, structurally intact sample.

These methods retain endogenous fluorescence and facilitate antibody penetration, making them ideal for multiplexed imaging. However, clearing can take days to weeks depending on tissue size. Active clearing techniques, such as electrophoretic tissue clearing (ETC), accelerate lipid removal but require specialized equipment. Despite these challenges, hydrogel-based methods are widely used in neuroscience and immunology for detailed molecular mapping of intact tissues.

Aqueous-Based

Aqueous-based clearing methods use high-refractive-index solutions to reduce light scattering without extensive lipid removal. Techniques such as Scale, CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), and SeeDB (See Deep Brain) rely on water-soluble agents like urea, fructose, or aminoalcohols to achieve transparency. These methods preserve fluorescence signals and minimize tissue shrinkage or expansion.

CUBIC, for example, employs aminoalcohols and detergents to penetrate deep tissues while preserving protein integrity, making it suitable for whole-organ imaging. Scale, which uses urea-based solutions, is effective for soft tissues like the brain but may cause swelling. Aqueous-based methods are generally more biocompatible than solvent-based approaches, making them preferable for long-term imaging studies. However, clearing efficiency can be lower, particularly for lipid-rich tissues, requiring extended incubation times.

Transparency Steps

Achieving uniform transparency requires precise control over each phase of the process. The transition from opaque to fully cleared tissue depends on the systematic removal or modification of light-scattering components, primarily lipids and water, while maintaining structural fidelity. Selecting reagents that match the tissue’s refractive index ensures seamless light transmission and improved resolution.

The refractive index mismatch between tissues and surrounding media causes optical distortion. Addressing this requires gradual adjustment of the sample’s refractive properties, often through immersion in clearing solutions with progressively increasing refractive indices. This stepwise approach prevents abrupt osmotic changes that could lead to tissue shrinkage or expansion. High-refractive-index solutions, such as iohexol- or glycerol-based mixtures, fine-tune transparency while minimizing structural deformation.

Lipid removal plays a central role in enhancing tissue clarity, particularly in lipid-rich organs. The efficiency of this process depends on both the chemical properties of the clearing agents and the duration of incubation. Amphipathic detergents like SDS disrupt lipid bilayers while preserving protein structures, allowing deep tissue penetration of optical clearing solutions. The concentration and exposure time must be carefully optimized to prevent excessive protein loss, which can weaken tissue integrity and reduce fluorescence signal retention.

Staining Sequence Considerations

The order of staining significantly impacts imaging quality in cleared tissues. Fluorescent markers, antibodies, and dyes must effectively penetrate the sample while preserving signal intensity and specificity. The choice of staining sequence depends on epitope accessibility, tissue permeability, and the potential for fluorescence quenching during clearing.

Tissue clearing alters the sample’s physical and chemical properties, affecting reagent diffusion. In some protocols, staining occurs before clearing to take advantage of the native antigenic state, reducing the risk of epitope masking. This approach benefits immunolabeling with large antibodies, which may struggle to penetrate fully cleared tissues. However, lipids can hinder pre-clearing staining, requiring extended incubation or permeabilization agents. Post-clearing staining is often preferred for smaller fluorophores or dyes that can diffuse efficiently through transparent tissues, ensuring uniform distribution.

Microscope Setup And Imaging

After clearing and staining, careful imaging setup is essential to maximize resolution and signal fidelity. The choice of microscope depends on sample size, imaging depth, and fluorescent labels. Confocal and two-photon microscopy are frequently used for cleared tissues due to their ability to capture high-resolution optical sections while minimizing photobleaching. Light-sheet fluorescence microscopy (LSFM) has gained prominence for whole-organ imaging, as it enables rapid volumetric acquisition with reduced phototoxicity.

Matching the refractive index of the imaging medium to that of the cleared tissue minimizes spherical aberrations, which can distort image quality. Many clearing protocols specify compatible immersion media, such as silicone oils or refractive index-matched aqueous solutions, to optimize light transmission. Objective lenses with long working distances and high numerical apertures are preferred for deep-tissue imaging. Autofluorescence reduction techniques, such as spectral unmixing or background subtraction, further enhance signal clarity.

Storage After Clearing

Proper storage conditions prevent sample degradation, fluorescence signal loss, and changes in transparency. The choice of storage medium should be compatible with the clearing method and fluorophores to ensure structural stability. Refractive index-matching solutions such as sRIMS (sorbitol-based refractive index matching solution) or fructose-based media help maintain optical clarity and prevent dehydration.

Cleared tissues should be stored in light-protected containers at 4°C to reduce photobleaching and oxidative damage. Some protocols recommend antifungal or antimicrobial additives to prevent microbial growth. For long-term preservation, embedding the sample in a hydrogel matrix or sealing it in an airtight chamber can prevent structural changes. Regular monitoring of transparency and fluorescence intensity ensures samples remain viable for future imaging sessions.