MIT expansion microscopy allows clearer views of brain tissue

15 Oct 2024

New protocol brings technique within reach of conventional labs and existing microscopes.

Expansion microscopy (ExM) is an attractive route to nanoscale imaging for labs without conventional super-resolution microscopes.

It involves linking fluorescent markers through a gel infused into the sample, which remains in place after the tissue itself is digested away.

The gel can then be expanded to create an enlarged phantom of the original sample. The relative distribution of fluorescent tags is maintained, but the expansion operation moves them sufficiently further apart that they can now be optically resolved.

Previous applications of ExM have included the 2023 study at Carnegie Mellon University which aimed to widen ExM's applicability through modifications to the gel employed, commenting that ExM still involved some "longstanding challenges."

A project at MIT has now tackled those challenges further, by developing a ExM protocol capable of achieving a 20-fold expansion in a single shot. The findings were published in Nature Methods.

"To date, ExM methods either expand specimens to a limited range (4–10× linearly), or achieve larger expansion factors through iterating the expansion process a second time to 15–20× linearly," commented the project in its paper. "We present an ExM protocol that achieves 20× expansion, yielding <20-nanometer resolution on a conventional microscope, in a single expansion step."

At the 20-nanometer resolution achieved by this technique scientists can see organelles inside cells and clusters of proteins, providing valuable data about nanoscale structures within the tissues.

"Twenty-fold expansion gets you into the realm that biological molecules operate in," commented Ed Boyden leader of the MIT Synthetic Neurobiology Group and also one of the pioneers of optogenetics. "The building blocks of life are nanoscale things: biomolecules, genes, and gene products."

Democratizing super-resolution imaging

The trick to 20-fold expansion was to find a gel that was extremely absorbent and mechanically stable enough that it would not fall apart when experiencing the enhanced levels of enlargement.

MIT's answer was a gel assembled from N,N-dimethylacrylamide (DMAA) and sodium acrylate. 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, said the researchers.

These gel components had been used previously in expansion microscopy protocols, but those gels could expand only about tenfold, so the MIT team optimized the gel and the polymerization process to make the gel more robust.

In trials of the new protocol, christened 20ExM, the project was able to image synaptic nanocolumns within brain cells, the clusters of proteins arranged in a specific way at synapses that allow neurons to communicate with each other via secretion of neurotransmitters.

Applying the new method to cancer cells, the project saw the hollow microtubules that help give cells their structure and play important roles in cell division. It also imaged mitochondria and the organization of individual nuclear pore complexes, the clusters of proteins that control access to the cell nucleus.

MIT hopes that any biology lab should be able to use this technique to approach super-resolution imaging performance, since it relies on standard off-the-shelf chemicals and common confocal microscopy equipment. Although more sample preparation may be involved compared to other super-resolution techniques, the actual imaging process is much more straightforward.

"This democratizes imaging," commented MIT's Laura Kiessling. "This new technique drives down the cost of imaging because you can see nanoscale things without the need for a specialized facility."