Scientists at Scripps Research report that they have unveiled a new tissue-clearing method for rendering large biological samples transparent. The method makes it much easier for scientists to visualize and study healthy and disease-related biological processes occurring across multiple organ systems, according to the researchers.

The team published a paper (“HYBRiD: hydrogel-reinforced DISCO for clearing mammalian bodies”) in Nature Methods and dubbed the method HYBRiD, which combines elements of the two main prior approaches to tissue-clearing technology, and should be more practical and scalable than either for large-sample applications, added the scientists.

“The recent development of solvent- and polymer-based brain-clearing techniques has advanced our ability to visualize the mammalian nervous system in three dimensions. However, it remains challenging to image the mammalian body en bloc. Here we developed HYBRiD (hydrogel-based reinforcement of three-dimensional imaging solvent-cleared organs (DISCO)), by recombining components of organic- and polymer-based clearing pipelines,” the investigators wrote.

“We achieved high transparency and protein retention, as well as compatibility with direct fluorescent imaging and immunostaining in cleared mammalian bodies. Using parvalbumin- and somatostatin-Cre models, we demonstrated the utility of HYBRiD for whole-body imaging of genetically encoded fluorescent reporters without antibody enhancement of signals in newborn and juvenile mice. Using K18-hACE2 transgenic mice, HYBRiD enabled perfusion-free clearing and visualization of SARS-CoV-2 infection in a whole mouse chest, revealing macroscopic and microscopic features of viral pathology in the same sample.

“HYBRiD offers a simple and universal solution to visualize large heterogeneous body parts or entire animals for basic and translational research.”

“This is a simple and universal tissue-clearing technique for studies of large body parts or even entire animals,” said study senior author Li Ye, PhD, assistant professor of neuroscience at Scripps Research.

Tissue clearing involves the use of solvents to remove molecules that make tissue opaque (such as fat), rendering the tissue optically transparent, while keeping most proteins and structures in place. Scientists commonly use genetically encoded or antibody-linked fluorescent beacons to mark active genes or other molecules of interest in a lab animal, and tissue clearing in principle allows these beacons to be imaged all at once across the entire animal.

Development of tissue-clearing techniques began about 15 years ago

Scientists started developing tissue-clearing methods about 15 years ago, mainly for the purpose of tracing nerve connections within whole brains. While the methods work well for brains, they don’t work so well when applied to other body parts or whole bodies, which contain harder-to-dissolve structures, noted Ye.

These methods until now have used either organic solvents or water-based solvents. The former generally work more quickly and powerfully but tend to diminish fluorescent signals. Methods using water-based solvents are better at preserving fluorescence but are impractically weak for clearing non-brain tissue. In addition, both types of methods require labor-intensive procedures, often using hazardous chemicals.

“An ordinary lab generally can’t use these methods routinely and at scale,” said Yu Wang, a graduate student in the Ye laboratory who was co-first author of the paper.

The new method devised by Ye and his team uses a sequential combination of organic solvents and water-based detergents, and makes use of water-based hydrogels to protect those molecules within the tissue that need to be preserved. It often does not require the pumping of solvents through the sample.

“In many cases, you can just put the whole thing in a jar and keep it in a shaker on your benchtop until it’s done,” said co-first author Victoria Nudell, a research assistant in the Ye lab. “This makes it practical and scalable enough for routine use.”

The researchers demonstrated the utility of their new method in a variety of applications. These included a collaboration with the laboratory of John Teijaro, PhD, associate professor of immunology and microbiology, to image SARS-CoV-2-infected cells in the whole chests of mice for the first time, which enabled it to be done in a high-level biosafety facility where access to equipment is strictly limited.

Ye and his team are now working with their scientific collaborators on multiple applications of the new method, including the tracing of nerve pathways in the body.

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