Expansion technique to image nanoscale structures inside cells makes high-resolution imaging more accessible

A classic way to image nanoscale structures in cells is to use expensive, high-powered super-resolution microscopes. As an alternative, MIT researchers have developed a way to expand tissues before imaging them, a technique that allows them to achieve nanoscale resolution with a conventional optical microscope.

In the latest version of this technique, researchers have made it possible to multiply tissues by 20 in a single step. This simple and inexpensive method could pave the way for almost all biology laboratories to perform nanoscale imaging.

“It democratizes imaging,” says Laura Kiessling, Novartis Professor of Chemistry at MIT and a member of the Broad Institute of MIT and Harvard and the Koch Institute for Integrative Cancer Research at MIT.

“Without this method, if you want to see things with high resolution, you have to use very expensive microscopes. This new technique allows you to see things that you normally couldn’t see with standard microscopes. the cost of imaging, because you can see objects at the nanoscale without the need for a specialized facility.

At the resolution achieved by this technique, which is around 20 nanometers, scientists can see organelles inside cells, as well as clumps of proteins.

“A twenty-fold expansion takes you into the realm in which biological molecules operate. The building blocks of life are nanoscale things: biomolecules, genes and gene products,” explains Professor Y Edward Boyden. Eva Tan in neurotechnology at MIT; professor of biological engineering, media arts and sciences, and brain and cognitive sciences; an investigator from the Howard Hughes Medical Institute; and a member of the McGovern Institute for Brain Research at MIT and the Koch Institute for Integrative Cancer Research.

Boyden and Kiessling are lead authors of the new study, which appears in Natural methods. MIT graduate student Shiwei Wang and Tay Won Shin Ph.D. are lead authors of the paper.

Only one extension

Boyden’s lab invented expansion microscopy in 2015. The technique requires embedding tissues in an absorbent polymer and breaking down the proteins that normally hold the tissues together. When water is added, the gel swells and separates the biomolecules from each other.

The original version of this technique, which quadrupled tissues, 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 stage, allowing for an overall 20-fold expansion. This allows for even higher resolution, but the process is more complicated.

“We have developed several 20-fold expansion technologies in the past, but they require multiple stages of expansion,” says Boyden. “If you could do such an expansion in one step, it would simplify things a little.”

With a 20-fold expansion, researchers can achieve a resolution of around 20 nanometers using a conventional optical microscope. This allows them to see cellular structures like microtubules and mitochondria, as well as protein clusters.

In the new study, the researchers decided to increase the volume 20-fold in a single step. This meant they had to find a gel that was both extremely absorbent and mechanically stable, so that it wouldn’t fall apart when multiplied by 20.

To achieve this, they used a gel assembled from N,N-dimethylacrylamide (DMAA) and sodium acrylate. Unlike previous expansion gels that relied on the addition of another molecule to form cross-links between polymer strands, this gel spontaneously forms cross-links and exhibits strong mechanical properties.

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

To further stabilize the gel and improve its reproducibility, the researchers removed oxygen from the polymer solution before gelation, which avoids side reactions that interfere with cross-linking. This step requires passing nitrogen gas through the polymer solution, which replaces most of the oxygen in the system.

Once the gel is formed, some bonds in the proteins that hold the tissues together are broken and water is added to expand the gel. Once expansion is complete, target proteins in the tissues can be labeled and imaged.

“This approach may require more sample preparation compared to other super-resolution techniques, but it is much simpler when it comes to the actual imaging process, especially for 3D imaging,” Shin explains. “We document the protocol step-by-step in the manuscript so readers can walk through it easily.”

Imaging tiny structures

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

In their studies of cancer cells, the researchers also imaged microtubules, hollow tubes that help give cells their structure and play an important role in cell division. They were also able to observe mitochondria (organelles that generate energy) and even the organization of individual nuclear pore complexes (clusters of proteins that control access to the cell nucleus).

Wang is now using this technique to image carbohydrates called glycans, which are found on the surface of cells and help control the cells’ interactions with their environment. This method could also be used to image tumor cells, allowing scientists to gain insight into how proteins are organized within these cells, much more easily than before.

The researchers envision that any biology laboratory should be able to use this technique at low cost because it relies on standard, commercially available chemicals and common equipment such as confocal microscopes and glove bags, of which most laboratories already have or can easily access.

“We hope that with this new technology, any conventional biology laboratory will be able to use this protocol with their existing microscopes, allowing them to approach a resolution that can only be achieved with very specialized, state-of-the-art microscopes and expensive,” Wang said. .

This story is republished courtesy of MIT News (web.mit.edu/newsoffice/), a popular site that covers news in MIT research, innovation and education.