Tissue clearing is a technique in biological imaging that renders opaque biological tissues transparent. This method allows researchers to visualize intricate structures in three dimensions. It provides a detailed view into the architecture of biological systems, from cellular networks to entire organs.
The Importance of Tissue Clearing
Traditional microscopy often relies on slicing tissues into very thin, two-dimensional sections, typically around 10 micrometers thick. This process destroys the three-dimensional context of the tissue, making it challenging to trace structures like blood vessels or nerve fibers over long distances. Reconstructing a complete 3D picture from numerous 2D slices is time-consuming and can introduce inaccuracies.
Tissue clearing addresses these limitations. It preserves the spatial relationships and connectivity of cells and structures within the sample. By allowing light to penetrate deeply, it enables comprehensive volumetric imaging, providing a deeper understanding of biological organization difficult to achieve with traditional sectioning methods.
How Tissue Clearing Works
The principle behind tissue clearing involves addressing two factors that cause tissues to be opaque: light scattering and light absorption. Light scattering occurs when light encounters components within the tissue that have different refractive indices, such as lipids, proteins, and water. These differences cause light rays to bend and disperse, making the tissue appear cloudy.
Clearing protocols primarily work by either removing lipids, which are significant light scatterers, or by homogenizing the refractive index throughout the tissue. For instance, some methods use detergents to extract lipids, while others immerse the tissue in solutions that have a refractive index similar to that of the remaining tissue components. This equalization allows light to pass through the sample with minimal disturbance.
Common Tissue Clearing Approaches
Various tissue clearing protocols have been developed.
Solvent-Based Methods
This category includes methods that typically involve dehydrating the tissue and then immersing it in organic solvents. These solvents remove lipids and simultaneously match the refractive index of the tissue. Examples include 3DISCO and iDISCO, which are known for their rapid clearing capabilities but can sometimes lead to tissue shrinkage and quenching of fluorescent proteins.
Hydrogel-Based Methods
Another approach involves cross-linking the tissue’s proteins to a hydrogel scaffold, maintaining structural integrity. Lipids are then extracted using detergents, and the tissue-hydrogel hybrid is immersed in a refractive index-matching solution. Protocols like CLARITY, PACT, and SHIELD fall into this category, offering good clearing performance and preservation of proteins, RNA, and DNA.
Aqueous-Based Methods
A third category relies on water-soluble solutions containing high refractive index molecules like sucrose, fructose, or glycerol. These methods are generally simpler and less hazardous than solvent-based techniques, and they are often compatible with fluorescent proteins. Examples include CUBIC, Scale, and SeeDB, though they can be slower for larger samples and some may cause tissue expansion.
Insights from Cleared Tissues
Tissue clearing has advanced biological discovery across various fields.
Neuroscience: It has enabled detailed mapping of neuronal networks and brain connectivity, allowing researchers to visualize entire neural circuits without sectioning. This capability is advancing understanding of neurological diseases and brain function.
Developmental Biology: This field benefits from observing organ formation and tissue growth in intact embryos, revealing complex spatial relationships during development. Researchers can now trace cell lineages and structural changes in three dimensions.
Cancer Research: Tissue clearing allows for comprehensive visualization of tumor vasculature, metastasis, and the tumor microenvironment. This helps understand how tumors grow, spread, and interact with surrounding tissues, which is valuable for developing new therapies.
Immunology: The technique aids in studying immune cell distribution and their interactions within their native tissue context.