MIT researchers have developed a new way to image proteins and RNA inside neurons of intact brain tissue. Image: Yosuke Bando, Fei Chen, Dawen Cai, Ed Boyden, and Young Gyu
Microscopy technique allows pinpointing RNA molecules in the brain. Key to the new technique is expanding the tissue before imaging it so it can be seen at very high resolution using ordinary microscopes commonly found in research labs. “Now we can image RNA with great spatial precision, thanks to the expansion process, and we also can do it more easily in large intact tissues,” says A/Prof Ed Boyden.
Studying the distribution of RNA inside cells could help scientists learn more about how cells control their gene expression and could also allow them to investigate diseases thought to be caused by failure of RNA to move to the correct location. Boyden and colleagues first described the underlying technique, known as expansion microscopy (ExM), last year, when they used it to image proteins inside large samples of brain tissue. MIT team has now presented a new version of the technology that employs off-the-shelf chemicals, making it easier for researchers to use.
The original expansion microscopy technique is based on embedding tissue samples in a polymer that swells when water is added. This tissue enlargement allows researchers to obtain images with a resolution of around 70nm. However, that method posed some challenges because it requires generating a complicated chemical tag consisting of an antibody that targets a specific protein, linked to both a fluorescent dye and a chemical anchor that attaches the whole complex to polyacrylate. Once the targets are labeled, the researchers break down the proteins that hold the tissue sample together, allowing it to expand uniformly as the polyacrylate gel swells.
In their new studies, to eliminate the need for custom-designed labels, the researchers used a different molecule to anchor the targets to the gel before digestion. This molecule, AcX can be modified to anchor either proteins or RNA to the gel. They used it to anchor proteins, and they also showed the technique works on tissue previously labeled with either fluorescent antibodies or proteins eg green fluorescent protein (GFP).
“This lets you use completely off-the-shelf parts, which means that it can integrate very easily into existing workflows,” Tillberg says. Using this approach, it takes about an hour to scan a piece of tissue 500 by 500 by 200 microns, using a light sheet fluorescence microscope. The researchers showed that this technique works for many types of tissues, including brain, pancreas, lung, and spleen.
In the Nature Methods paper, the researchers used the same kind of anchoring molecule but modified it to target RNA instead. All of the RNAs in the sample are anchored to the gel, so they stay in their original locations throughout the digestion and expansion process.
After the tissue is expanded, the researchers label specific RNA molecules using fluorescence in situ hybridization (FISH). This allows researchers to visualize the location of specific RNA molecules at high resolution, in 3D, in large tissue samples. This allows scientists to explore many questions about how RNA contributes to cellular function. eg a longstanding question in neuroscience is how neurons rapidly change the strength of their connections to store new memories or skills. One hypothesis is that RNA molecules encoding proteins necessary for plasticity are stored in cell compartments close to the synapses, poised to be translated into proteins when needed.
With the new system, it should be possible to determine exactly which RNA molecules are located near the synapses, waiting to be translated.
Boyden’s lab is also interested in using this technology to trace the connections between neurons and to classify different subtypes of neurons based on which genes they are expressing. http://news.mit.edu/2016/rna-nanoscale-brain-0704
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