See the brain as never before
Image: Expansion plus lattice light-sheet microscopy provides rapid 3D images of the brain
Nobel prize winner, Eric Betzig, and expansion microscopy pioneer, Ed Boyden, have teamed up to image the brain with incredible speed and nanoscale resolution.
By combining lattice sheet microscopy with expansion microscopy, Betzig, Boyden and colleagues have imaged an entire fruit fly brain in only 62.5 hours, showing details as small as 60 nm.
Fly brain: the coloured balls indicate the density of synapses on a subset of the neurons in the brain, that respond to dopamine. The balls summarise the placement of a total of 500,000 synapses, out of 40 million in the entire brain, where red indicates the highest synapse density, purple the lowest. [HHMI/UC Berkeley]
The incredibly detailed 3D map of the brain is assembled from millions of 2D images and comprises some 40 million synapses.
While the level of detail doesn't quite match that obtained with an electron microscope, efforts to fully map the neurons and synapses of the fly brain with electron microscopy have taken decades, with the efforts of dozens of people.
As Ed Boyden, from MIT, and a HHMI researcher, points out: "Optical microscopy doesn't offer sufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualise specific molecular constituents over millimetre-scale or larger dimensions."
"[But with] combined expansion microscopy and lattice light-sheet microscopy, we've crossed a threshold in imaging performance," he adds. "That's why we're so excited. We're not just scanning incrementally more brain tissue, we're scanning entire brains."
After expanding the fruit fly brain to four times its usual size, scientists used lattice light-sheet microscopy to image all of the dopaminergic neurons (green). [Gao et al./Science 2019]
As Betzig from UC Berkeley and a HHMI researcher recalls, when Boyden and his team approached him to ask if they could use his lattice sheet microscope to swiftly and gently image large brain samples - expanded to four times their usual size - he wasn't impressed.
"I thought they were full of it and I was going to show them," he laughs.
"But then you could have knocked me over with a feather... I couldn't believe the quality of the data I was seeing," he adds.
A forest of dendritic spines protrude from the branches of neurons in the mouse cortex. [Gao et al./ Science 2019]
Expansion microscopy involves infusing neural tissue with polyacrylamide gel and then swelling it, while keeping the relative positions of internal structures unchanged.
Meanwhile, lattice light-sheet microscopy uses highly focused light beams to rapidly assemble a 3D image of a specimen one thin slice at a time.
Combined, the technique allows researchers to map large-scale circuits within brains, imaging the nanoscale spatial relationships between proteins across the thickness of the mouse cortex as well as the entire Drosophila brain.
Researchers can identify organelles of various shapes and sizes (coloured areas) inside mouse neurons imaged by a technique that merges expansion microscopy with lattice light-sheet microscopy. [Gao et al./ Science 2019]
"A lot of problems in biology are multiscale," says Boyden. "Using lattice light-sheet microscopy, along with the expansion microscopy process, we can now image at large scale without losing sight of the nanoscale configuration of biomolecules."
According to the researchers, using this technique, they can analyse millions of synapses in just a few days.
"We counted clusters of postsynaptic markers across the [mouse brain] cortex, and we saw differences in synaptic density in different layers of the cortex," explains Ruixuan Gao, from Ed Boyden's MIT laboratory. "Using electron microscopy, this would have taken years to complete."
Indeed, as Betzig adds: "I can see us getting to the point of imaging at least ten fly brains per day."
What's more, Betzig reckons that with improvements in expansion microscopy, the combined techniques could achieve results nearly as good as electron microscopy, in terms of mapping all the neural connections in the brain.
"If you could get this to work at 10 times or maybe 15 times expansion, you could probably put a lot of electron microscopes out of business," he says. "[This method] might be good enough to perform the dense neural tracing that electron microscopy can do but only much much faster and cheaper... It is not there yet, but in my opinion, the potential is there."
Three individually traced neurons in the fruit fly brain project from the antenna lobe in a bundle [Gao et al./ Science 2019]
The researchers went on to study axon myelination in neurons, the disruption of which is a hallmark of multiple sclerosis.
They computed the thickness of the myelin coating in different segments of axons, and measured the gaps between stretches of myelin; this kind of myelin tracing typically take months to years to perform.
This method has also been used to image organelles inside neurons, with the researchers identifying variations in the shapes of mitochondria and lysosomes.
Still, as Betzig points out, challenges remain.
According to the Nobel laureate, as with any kind of super resolution fluorescence microscopy, it can be hard to decorate proteins with enough fluorescent bulbs to see them clearly at high resolution.
And since expansion microscopy requires many processing steps, there's still the potential for artefacts to be introduced.
"Because of this, we have worked very hard to validate what we've done, and others would be well advised to do the same," he says.
The researchers are now building a new lattice light-sheet microscope, which they plan to move to Boyden's lab at MIT.
"Our hope is to rapidly make maps of entire nervous systems," says Boyden.