Microscopy technique reveals hidden nanostructures

CAMBRIDGE, MA — Inside a living cell, proteins and other molecules are often tightly packed together. These dense clumps can be difficult to image because the fluorescent markers used to make them visible cannot get stuck between the molecules.

MIT researchers have now developed a new way to overcome this limitation and make these “invisible” molecules visible. Their technique allows them to “decongest” molecules by expanding a cell or tissue sample before labeling the molecules, making the molecules more accessible to fluorescent labels.

The method, which builds on a widely used technique known as expansion microscopy previously developed at MIT, should allow scientists to visualize molecules and cellular structures that have never been seen before.

“It is becoming clear that the expansion process will reveal many new biological discoveries. If biologists and clinicians have studied a protein in the brain or another biological specimen and label it in the usual way, they may be missing entire categories of phenomena, says Edward Boyden, Professor Y Eva Tan in neurotechnology, professor of biological engineering and brain and cognitive sciences at MIT, researcher at the Howard Hughes Medical Institute and a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research at MIT.

Using this technique, Boyden and his colleagues showed that they could image a nanostructure found in the synapses of neurons. They also imaged the structure of beta-amyloid plaques linked to Alzheimer’s disease in more detail than before.

“Our technology, which we have named expansion revelation, allows the visualization of these nanostructures, which previously remained hidden, using material readily available in university laboratories,” explains Deblina Sarkar, assistant professor at the Media Lab and one of the main authors of the study. .

The lead authors of the study are Boyden; Li-Huei Tsai, director of MIT’s Picower Institute for Learning and Memory; and Thomas Blanpied, professor of physiology at the University of Maryland. Other lead authors include Jinyoung Kang, a postdoctoral fellow at MIT, and Asmamaw Wassie, a recent PhD graduate at MIT. The study appears today in Nature Biomedical Engineering.

Decongestion

Imaging a specific protein or other molecule inside a cell requires labeling it with a fluorescent tag carried by an antibody that binds to the target. Antibodies are about 10 nanometers long, while typical cell proteins are usually about 2-5 nanometers in diameter, so if the target proteins are too dense, the antibodies cannot reach them.

This has been a barrier to traditional imaging as well as the original version of expansion microscopy, which Boyden first developed in 2015. In the original version of expansion microscopy, researchers attached Fluorescent labels to molecules of interest before expanding the tissue. Labeling was done first, in part because the researchers had to use an enzyme to chop up the proteins in the sample so the tissue could be expanded. This meant that proteins could not be labeled after tissue expansion.

To overcome this hurdle, the researchers had to find a way to expand the tissue while leaving the proteins intact. They used heat instead of enzymes to soften the tissue, allowing the tissue to expand 20 times without being destroyed. Then, the separated proteins could be tagged with fluorescent tags after expansion.

With so many additional proteins accessible for labeling, the researchers were able to identify tiny cellular structures in synapses, the connections between neurons that are densely packed with protein. They labeled and imaged seven different synaptic proteins, allowing them to visualize in detail “nanocolumns” made up of calcium channels aligned with other synaptic proteins. These nanocolumns, believed to help make synaptic communication more efficient, were first discovered by Blanpied’s lab in 2016.

“This technology can be used to answer many biological questions about the dysfunction of synaptic proteins, which are implicated in neurodegenerative diseases,” says Kang. “Until now, there was no tool to visualize synapses well.”

New patterns

The researchers also used their new technique to image beta-amyloid, a peptide that forms plaques in the brains of patients with Alzheimer’s disease. Using mouse brain tissue, the researchers discovered that beta-amyloid forms periodic nanoclusters, which had never been seen before. These beta-amyloid clusters also include potassium channels. The researchers also found beta-amyloid molecules that formed helical structures along axons.

“In this article, we do not speculate on what this biology might mean, but we show that it exists. This is just one example of the new patterns we can see,” says MIT graduate student Margaret Schroeder, who also authored the paper.

Sarkar says she is fascinated by the nanoscale biomolecular patterns that this technology unveils. “With a background in nanoelectronics, I have developed electronic chips that require extremely precise alignment, in the nanofab. But when I see that in our brain, Mother Nature has arranged biomolecules with such precision at the nanoscale, it really amazes me,” she says.

Boyden and members of his group are now working with other labs to study cellular structures such as protein aggregates linked to Parkinson’s disease and other diseases. In other projects, they study pathogens that infect cells and molecules involved in brain aging. Preliminary results from these studies have also revealed new structures, Boyden says.

“Time and time again you see things that are really shocking,” he says. “It shows us everything we miss with classic unexpanded coloring.”

The researchers are also working on modifying the technique so they can image up to 20 proteins at once. They are also working to adapt their process so that it can be used on human tissue samples.

Sarkar and his team, on the other hand, are developing tiny, wirelessly powered nanoelectronic devices that could be delivered to the brain. They plan to integrate these devices with a revealing extension. “It can combine the intelligence of nanoelectronics with the nanoscopy prowess of expansion technology, for an integrated functional and structural understanding of the brain,” says Sarkar.

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The research was funded by the National Institutes of Health, National Science Foundation, Ludwig Family Foundation, JPB Foundation, Open Philanthropy Project, John Doerr, Lisa Yang, and MIT’s Tan-Yang Center for Autism Research. US Army. Office of Research, Charles Hieken, Tom Stocky, Kathleen Octavio, Lore McGovern, Good Ventures and HHMI.


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