A classical way to image nanoscale structures in cells is with high-powered, expensive super-resolution microscopes. As an alternative, MIT researchers have developed a single-step technique for expanding tissue before imaging it, making it possible achieve nanoscale resolution with a conventional light microscope. At this resolution—around 20 nanometers— it’s possible to see organelles inside cells, as well as clusters of proteins.

The team suggests the simple, inexpensive method, which they’ve called 20ExM could pave the way for nearly any biology lab to perform nanoscale imaging. “Twenty-fold expansion gets you into the realm that biological molecules operate in,” said Edward Boyden, PhD, the Y. Eva Tan Professor in Neurotechnology at MIT. “The building blocks of life are nanoscale things: biomolecules, genes, and gene products.” Boyden is a professor of biological engineering, media arts and sciences, and brain and cognitive sciences; a Howard Hughes Medical Institute investigator; and a member of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research.

“This democratizes imaging,” added Laura Kiessling, PhD, the Novartis Professor of Chemistry at MIT and a member of the Broad Institute of MIT and Harvard and MIT’s Koch Institute for Integrative Cancer Research. “Without this method, if you want to see things with a high resolution, you have to use very expensive microscopes. What this new technique allows you to do is see things that you couldn’t normally see with standard microscopes. It drives down the cost of imaging because you can see nanoscale things without the need for a specialized facility.”

Kiessling and Boyden are co-senior authors of the team’s published paper in Nature Methods, titled “Single-shot 20-fold Expansion Microscopy Enables 18-nm Resolution Imaging on Conventional Microscopes,” in which they concluded “20ExM may find utility in many areas of biological investigation requiring high-resolution imaging.” The paper’s lead authors are MIT graduate student Shiwei Wang, and Tay Won Shin, PhD.

Identifying and locating biomolecules with nanoscale precision in intact cells and tissues is key to understanding their roles in such biological systems, the authors wrote. Boyden’s lab invented expansion microscopy in 2015. The technique requires embedding tissue into an absorbent polymer and breaking apart the proteins that normally hold tissue together. When water is added, the gel swells and pulls biomolecules apart from each other. “After the embedded specimens are chemically softened and the hydrogel is immersed in water, the polymer network expands isotropically while preserving the relative spatial organization of the anchored molecules,” the investigators further explained. “Expansion microscopy (ExM) provides a robust, simple, and affordable solution because its isotropic physical magnification enables nanoscale resolution imaging of preserved cells and tissues on conventional microscopes.”

The original version of ExM technology, which expanded tissue about fourfold, allowed researchers to obtain images with a resolution of around 70 nanometers. In 2017, Boyden’s lab modified the process to include a second expansion step, achieving an overall 20-fold expansion. This enables even higher resolution. With 20-fold expansion researchers can get down to a resolution of about 20 nanometers, using a conventional light microscope. This allows them see cell structures such as microtubules and mitochondria, as well as clusters of proteins. However, the process is more complicated. “Previous ExM methods either expanded specimens to a limited range in one shot (~4–10x linearly) or achieved higher expansion factors through re-embedding the first gel in a second hydrogel, and then iterating the expansion process again (~15–20x linear expansion total),” the team commented.

Boyden added, “We’ve developed several 20-fold expansion technologies in the past, but they require multiple expansion steps. If you could do that amount of expansion in a single step, that could simplify things quite a bit.”

For their newly reported work the researchers set out to perform 20-fold expansion with only a single step. This meant that they had to find a gel that was both extremely absorbent and mechanically stable so that it wouldn’t fall apart when expanded 20-fold. “… a polymer with exceptional mechanical properties is needed,” the investigators noted.

To achieve this the team used a gel assembled from N,N-dimethylacrylamide (DMAA) and sodium acrylate (SA). Unlike previous expansion gels that rely on adding another molecule to form crosslinks between the polymer strands, this gel forms crosslinks spontaneously and exhibits strong mechanical properties. “We chose to optimize a hydrogel composed of N,N-dimethylacrylamide (DMAA) and sodium acrylate (SA), reagents that are known to  form mechanically robust and elastic hydrogels, due to the unique self-crosslinking chemistry of DMAA,” they further stated.

Such gel components previously had been used in expansion microscopy protocols, but the resulting gels could expand only about tenfold. The MIT team optimized the gel and the polymerization process to make the gel more robust, and to allow for 20-fold expansion.

To further stabilize the gel and enhance its reproducibility, the researchers removed oxygen from the polymer solution prior to gelation, which prevents side reactions that interfere with crosslinking. This step requires running nitrogen gas through the polymer solution, which replaces most of the oxygen in the system.

Once the gel is formed, select bonds in the proteins that hold the tissue together are broken and water is added to make the gel expand. After the expansion is performed, target proteins in tissue can be labeled and imaged. “This approach may require more sample preparation compared to other super-resolution techniques, but it’s much simpler when it comes to the actual imaging process, especially for 3D imaging,” Shin said. “We document the step-by-step protocol in the manuscript so that readers can go through it easily.”

Using their technique, the researchers were able to image many tiny structures within brain cells, including structures called synaptic nanocolumns. These are clusters of proteins that are arranged in a specific way at neuronal synapses, allowing neurons to communicate with each other via secretion of neurotransmitters such as dopamine.

In studies of cancer cells the researchers imaged microtubules—hollow tubes that help give cells their structure and play important roles in cell division. They were also able to see mitochondria and even the organization of individual nuclear pore complexes, clusters of proteins that control access to the cell nucleus. “In one round of expansion, this protocol, which we call 20ExM, reveals hollow microtubule structures in cultured cells and synaptic nanocolumns in mouse somatosensory cortex, on a conventional confocal microscope,” the authors reported. “… 20ExM achieves a resolution comparable to iterative expansion methods (~20 nm) with a single expansion step.”

Wang is now using this technique to image carbohydrates known as glycans, which are found on cell surfaces and help to control cells’ interactions with their environment. This method could also be used to image tumor cells, allowing scientists to glimpse how proteins are organized within those cells, much more easily than has previously been possible.

The authors concluded. “We anticipate 20ExM to find broad utility in biology due to its high performance and simplicity … As demonstrated in both cell culture and tissue specimens, 20ExM can be immediately deployed in a wide variety of experimental contexts where high resolution and single-step simplicity are desired. 20ExM could, in principle, be used to simplify, and/or enhance the resolution of, other expansion-based technologies, such as in situ RNA detection and sequencing, genome imaging, multiplexed proteomics, and lipid and glycan staining.”

The researchers envision that any biology lab should be able to use this technique at a low cost since it relies on standard, off-the-shelf chemicals and common equipment such confocal microscopes and glove bags, which most labs already have or can easily access.

“Our hope is that with this new technology, any conventional biology lab can use this protocol with their existing microscopes, allowing them to approach resolution that can only be achieved with very specialized and costly state-of-the-art microscopes,” Wang said.

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