About 3D Tissue Clearing
Tissue clearing methodologies have emerged as powerful techniques in the histology space, enabling unprecedented insights into the three-dimensional structure of intact biological samples. While traditional histological techniques cut tissues into thin slices, tissue clearing is able to investigate intact tissues, providing an unbiased and complete view of biological samples.
To provide researchers with a window into intact tissues, tissue clearing methodologies render intact biological samples optically transparent by removing light-scattering lipids while preserving proteins for immunolabeling.
By immersing the cleared tissue in a solution with the same refractive index as the preserved proteins, diffraction, and light scattering are minimized. This allows light to pass through the sample, providing access to labeled proteins deep within the tissue. These transparent samples, coupled with Light Sheet Fluorescence Microscopy (LSFM) enable imaging of the entire intact sample to visualize the labeled proteins of interest.
Tissue clearing preserves the 3D spatial information and relationships within the sample– particularly valuable for studying complex three-dimensional structures, such as axon projections, vascular networks, and drug biodistribution, which can be challenging to visualize and quantify using conventional 2D slice methods.
One of the most widely adopted tissue clearing protocols is the iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) method. The iDISCO+ clearing and immunolabling methodology, combined with light sheet imaging, allows for high resolution 3D imaging of immunostained organs and tissues.
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Translucence Biosystems Modified iDISCO+ Protocol
At Translucence Biosystems, we employ a modified version of the iDISCO+ protocol co-developed by our Chief Technology Officer, Ricardo Azevedo. Prior to joining Translucence Biosystems, Azevedo contributed to the development of the original iDISCO+ protocol when in Marc Tessier-Levine’s lab at Rockefeller University.
The Translucence modified iDISCO+ protocol renders intact samples optically transparent while preserving their three-dimensional structure. This allows for image acquisition on any of the major commercially available light sheet microscopes.
Our protocol uses affordable, commercially available refractive index matching solutions and results in zero shrinkage of the samples of interest, making it cost-effective and compatible with downstream anatomical analysis.
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Summary
Unlike traditional 2D slice-histology methods, which rely on region of interest (ROI) slice selection, tissue clearing allows for robust analysis of the entire sample in a single workflow, eliminating potential biases and providing comprehensive and reliable data.
