Expansion microscopy blows brains up twice
Image: By expanding brain tissue twice, researchers obtained high-resolution images of neurons in the hippocampus.
MIT researchers have stretched the boundaries of expansion microscopy boosting image resolution from 60 nm to 25 nm.
The new technique, called iterative expansion microscopy and pioneered by Professor Ed Boyden and colleagues at MIT Media Lab, relies on expanding tissue twice and then imaging it with a conventional light microscope.
Two years ago, the MIT team showed that it was possible to expand tissue volumes 100-fold, resulting in an image resolution of about 60 nm.
Now, the researchers have shown that expanding the tissue a second time before imaging leads to a resolution of around 25 nm.
This level of resolution allows researchers to examine, for example, the proteins that cluster together in complex patterns at brain synapses, helping neurons to communicate with each other.
And as Boyden highlights, the greater resolution could also help researchers to map neural circuits.
“We want to be able to trace the wiring of complete brain circuits,” he says. “If you could reconstruct a complete brain circuit, maybe you could make a computational model of how it generates complex phenomena like decisions and emotions."
"Since you can map out the biomolecules that generate electrical pulses within cells and exchange chemicals between cells, you could potentially model the dynamics of the brain,” he adds.
To expand tissue, the researchers embed samples in a dense, absorbent, polyacrylate gel, used in nappies.
Before the gel is formed, the researchers label the cell proteins they want to image with antibodies that bind to specific targets.
These antibodies bear “barcodes” made of DNA, which in turn are attached to cross-linking molecules that bind to the polymers that make up the expandable gel.
The researchers then break down the proteins that normally hold the tissue together, allowing the DNA barcodes to expand away from each other as the gel swells.
These enlarged samples can then be labeled with fluorescent probes that bind the DNA barcodes, and imaged with commercially available confocal microscopes.
But as Boyden points out: “Individual biomolecules are... say 5 nanometres or even smaller... so the original versions of expansion microscopy were useful for many scientific questions but couldn’t equal the performance of the highest-resolution imaging methods such as electron microscopy.”
In past research, the researcher could expand tissue more than 100-fold in volume by reducing the number of cross-linking molecules that hold the polymer in an orderly pattern.
Still, according to Boyden: “If you reduce the cross-linker density, the polymers no longer retain their organization during the expansion process; you lose the information.”
Given this, the researchers modified their technique so that after the first tissue expansion, they create a new gel that swells the tissue a second time; an approach called 'iterative expansion'.
In this way, the researchers were able to image tissues with a resolution of about 25 nm, which is similar to that achieved by high-resolution techniques such as stochastic optical reconstruction microscopy (STORM).
Crucially, expansion microscopy is much cheaper and simpler to perform as specialised equipment or chemicals are not required.
Using iterative expansion, the researchers imaged synapses in finer details, identifying, for example, the location of neurotransmitter receptors on the surfaces of so-called postsynaptic cells on the receiving side of the synapse.
"My hope is that we can, in the coming years, really start to map out the organization of these scaffolding and signaling proteins at the synapse,” says Boyden.
The researcher also hopes to combine expansion microscopy with a new tool called temporal multiplexing, in which researchers can label one molecule with a fluorescent probe, take an image, and then wash the probe away.
This can be repeated many times, each time using the same colours to label different molecules.
“By combining iterative expansion with temporal multiplexing, we could in principle have essentially infinite-colour, nanoscale-resolution imaging over large 3D volumes,” highlights Boyden. “Things are getting really exciting now that these different technologies may soon connect with each other.”
The researchers also hope to achieve a third round of expansion, which they believe could, in principle, enable a resolution of about 5 nm.
Research is published in Nature Methods.
Learn more about Professor Ed Boyden here.