Tissue clearing is a chemical process that renders opaque biological samples, such as entire organs or large tissue blocks, optically transparent for microscopic examination. This technique was developed to overcome the limitations of traditional histology, where samples must be cut into extremely thin, two-dimensional slices before they can be imaged. Slicing tissue inevitably disrupts the intricate three-dimensional context of cellular networks and anatomical connections. By making the tissue transparent, researchers can use specialized volumetric microscopes, like light-sheet microscopy, to visualize and map complex structures throughout the intact sample. This approach provides an unbiased, system-level view of biological architecture.
The Science of Transparency
The natural opacity of biological tissue stems primarily from light scattering, which occurs when light rays are deflected as they pass through the sample. This scattering is caused by the difference in the refractive index (RI) between the various components within the tissue. A refractive index measures how much light is bent as it passes through a medium.
For example, the aqueous cytosol inside cells has an RI close to water, approximately 1.33, while the lipid membranes and protein components have RIs ranging from about 1.45 to 1.6. These abrupt boundaries between materials with different RIs cause light to scatter many times, preventing deep light penetration and making the tissue appear cloudy. Tissue clearing works by minimizing these inhomogeneities, a process known as refractive index matching (RIM).
The clearing solutions replace the water and lipids with a medium whose RI closely matches the RI of the remaining preserved proteins and nucleic acids. By creating a chemically uniform environment with a consistent RI across the entire volume, light can pass through the sample virtually undisturbed. This chemical manipulation effectively removes the physical basis for light scattering, allowing imaging light to penetrate to depths of several millimeters with high resolution.
Essential Steps in Tissue Clearing
Any tissue clearing protocol begins with sample preparation, which typically involves fixation to stabilize the tissue structure. Perfusion with a chemical fixative, such as paraformaldehyde, is often used to cross-link proteins and prevent tissue degradation during the subsequent chemical processing steps. Adequate fixation is necessary to maintain the physical integrity of the tissue, ensuring that the morphology of delicate structures is preserved throughout the protocol.
Following fixation, labeling or staining makes the structures of interest visible. If the sample does not naturally contain fluorescent proteins, researchers introduce molecular probes, such as fluorescently tagged antibodies, to bind to specific targets. For these large molecules to penetrate the dense tissue, the sample must be made permeable, a process that is often concurrent with the next step.
The delipidation or permeabilization step removes the major light-scattering components, primarily lipids, from the tissue. Detergents or organic solvents are used to dissolve and wash away these lipid membranes, which increases the transparency of the sample and allows solutions to penetrate deeper. This action also ensures that the protein structures are more accessible to the final clearing medium.
The final step is the refractive index matching (RIM), where the sample is immersed in the designated clearing medium. This medium is formulated to have an RI that matches the average RI of the remaining tissue components, thus creating the optical homogeneity required for transparency. Once the tissue is fully infiltrated with this final solution, the sample is ready for three-dimensional imaging.
Comparing Major Clearing Techniques
The many available tissue clearing protocols can be broadly categorized into three groups based on the chemical strategies they employ to achieve transparency.
Solvent-Based Methods
These methods, such as 3DISCO, rely on organic solvents for both delipidation and RIM. These protocols are known for their speed and ability to clear large, dense tissues quickly, often within a few days. However, the use of harsh organic solvents can lead to significant tissue shrinkage and often quenches the signal from endogenous fluorescent proteins, requiring additional signal amplification steps.
Aqueous-Based Methods
This approach includes protocols like Scale and CUBIC, which use water-soluble compounds like high concentrations of sugar or urea. These hyper-hydrating protocols are gentler on the tissue and offer excellent preservation of fluorescent proteins, making them advantageous for samples with genetically encoded reporters. A drawback is that they are generally slower and less effective at clearing very large, dense samples, and they can sometimes cause the tissue to expand.
Hydrogel-Based Methods
Exemplified by the CLARITY and PACT techniques, these methods focus on maintaining structural integrity. The tissue is first embedded in a stabilizing hydrogel scaffold, which cross-links the proteins before the lipids are chemically removed using detergents. This mechanical support prevents the structural degradation, shrinkage, or expansion that can occur during the aggressive delipidation process. While hydrogel methods are technically more complex and can be slow, they are unparalleled in their ability to preserve the tissue’s physical structure and allow for deep molecular interrogation.
Primary Research Applications
Tissue clearing has applications across numerous fields:
- Neuroscience: It enables the visualization of entire neural circuits and the comprehensive mapping of cell distribution across whole mouse brains. This capability allows researchers to trace long-range axonal projections and examine the complex architecture of the connectome without sectioning.
- Oncology: The technique provides a way to visualize the entire tumor microenvironment in three dimensions, offering a more complete understanding of tumor boundaries, vascularization, and immune cell infiltration. By imaging the intact tissue, scientists can analyze the spatial relationships between different cell types in the cancer ecosystem.
- Developmental Biology: Clearing allows for the tracking of organ formation and the analysis of developmental processes in intact embryos. This ability to obtain an unbiased, system-level view has expanded the scope of structural analysis across numerous organ systems, including the lung, kidney, and calcified tissues like bone.