Karolinska Institutet improves optical clearing for better views of brains

26 Nov 2024

New protocol allows 3D RNA imaging of whole mouse brains without sectioning.

Optical clearing, whereby biological tissues are rendered transparent to visible light, is a valuable route to imaging of cells and organs in their complete environment.

Multiple variations of tissue-clearing protocol have been developed, often involving a replacement of the extracellular material surrounding cells with a different substance of higher refractive index.

To date most of these protocols have been designed to allow assessment of how proteins are distributed in tissues; valuable data for sure, but not the only thing that biologists are keen to know.

One particular area of interest is the imaging of RNA in the tissues. Recent progress in this area includes the 2020 project at TU Wien that developed a clearing protocol specifically designed to retain compatibility with clinical fluorescence RNA imaging. But overall RNA imaging has remained challenging.

A project at Sweden's Karolinska Institutet and Karolinska University Hospital has now developed a microscopy method designed to show where RNA is expressed at a cellular level, and reported the work in Science.

"Recent advances in RNA analysis have deepened our understanding of cellular states in biological tissues," commented the project in its published paper. "However, a substantial gap remains in integrating RNA expression data with spatial context across organs, primarily owing to the challenges associated with RNA detection within intact tissue volumes."

Complex anatomical structures revealed

The project's new solution is TRISCO, named from Tris buffer–mediated retention of in situ hybridization chain reaction signal in cleared organs.

Earlier work at Karolinska Institutet had studied how an optical clearing technique using organic solvents followed by a hybridization chain reaction method could attach suitable fluorescent markers to the RNA molecules of interest, but the technique was limited to brain sections and biopsy samples.

"This method was unsuitable for visualizing RNA across intact brains owing to issues with poor RNA integrity, inhomogeneous labeling, and weak transparency," commented the researchers.

TRISCO aimed to improve these factors, including improved transparency of whole brain samples through adjustments to the methodology commonly used. A step involving treatment with a saline-sodium citrate buffer was eliminated, once the project found that washing the brains with water instead brought about a substantially improved transparency.

In trials using diverse organs of varying sizes and species, TRISCO proved able to achieve whole-brain RNA imaging at single-cell resolution, as well as identify landmarks of the brain such as the neocortex and hippocampus, without the need to slice the organ into sections.

The Karolinska Institutet team now believes that TRISCO may be suitable not only for studying intact mouse brains, but can be used for larger brains such as those of guinea pigs, as well as various tissues like kidney, heart, and lung.

"This method is a powerful tool that can drive brain research forward," commented Per Uhlén from Karolinska's Department of Medical Biochemistry and Biophysics. "With TRISCO, we can study the complex anatomical structure of the brain in a way that was previously not possible."