A forest of dendritic spines protrude from the branches of neurons in the mouse cortex.
Credit: Gao et al./ Science 2019
Two scientists, Ruixuan Gao and Shoh Asano, wanted to use his team's
microscope on brain samples expanded to four times their usual size --
blown up like balloons. The duo, part of Howard Hughes Medical Institute
(HHMI) Investigator Ed Boyden's lab at the Massachusetts Institute of
Technology (MIT), uses a chemical technique to make small specimens
bigger so scientists can more easily see molecular details.
Their technique, called expansion microscopy, worked well on single
cells or thin tissue sections imaged in conventional light microscopes,
but Boyden's team wanted to image vastly larger chunks of tissue. They
wanted to see complete neural circuits spanning millimeters or more. The
scientists needed a microscope that was high-speed, high resolution,
and relatively gentle -- something that didn't destroy a sample before
they could finish imaging it.
So, they turned to Betzig. His team at HHMI's Janelia Research Campus
had used their lattice light-sheet microscope to image the rapid
subcellular dynamics of sensitive living cells in 3-D. Combining the two
microscopy techniques could potentially offer rapid, detailed images of
wide swaths of brain tissue.
"I thought they were full of it," Betzig remembers. "The idea does
sound a bit crude," Gao says. "We're stretching tissues apart." But
Betzig invited Gao and Asano to try the lattice scope out.
"I was going to show them," Betzig laughs. Instead, he was blown
away. "I couldn't believe the quality of the data I was seeing. You
could have knocked me over with a feather."
Now, he and his Janelia colleagues have teamed up with Boyden's group
and imaged the entire fruit fly brain and sections of mouse brain the
thickness of the cortex. Their combined method offers high resolution
with the ability to visualize any desired protein -- and it's fast, too.
Imaging the fly brain in multiple colors took just 62.5 hours, compared
to the years it would take using an electron microscope, Boyden,
Betzig, and their colleagues report January 17, 2018, in the journal, Science.
"I can see us getting to the point of imaging at least 10 fly brains
per day," says Betzig, now an HHMI investigator at the University of
California, Berkeley. Such speed and resolution will let scientists ask
new questions, he says, like how brains differ between males and
females, or how brain circuits vary between flies of the same type.
Boyden's group dreams of making a map of the brain so detailed you
can simulate it in a computer. "We've crossed a threshold in imaging
performance," he says. "That's why we're so excited. We're not just
scanning incrementally more brain tissue, we're scanning entire brains."
Expanding the brain
Making detailed maps of the brain requires charting its activity and
wiring -- in humans, the thousands of connections made by each of more
than 80 billion neurons. Such maps could help scientists spot where
brain disease begins, build better artificial intelligence, or even
explain behavior. "That's like the holy grail for neuroscience," Boyden
says.
Years ago, his group had an idea to figure out how everything was
organized: What if they could actually make the brain bigger -- big
enough to look inside? By infusing samples with swellable gels -- like
the stuff in baby diapers -- the team invented a way to expand tissues,
making the molecules inside less crowded and easier to see under a
microscope. Molecules lock into a gel scaffold, keeping the same
relative positions even after expansion.
But it wasn't easy to image large tissue volumes. The thicker a
specimen gets, the harder it is to illuminate only the parts you want to
see. Shining too much light on samples can photobleach them, burning
out the fluorescent "bulbs" scientists use to light up cells.
Expanding a sample just four-fold increases its volume 64-fold, so
imaging speed also becomes paramount, Gao says. "We needed something
that was fast and didn't have much photobleaching, and we knew there was
a fantastic microscope at Janelia."
The lattice light-sheet microscope sweeps an ultrathin sheet of light
through a specimen, illuminating only that part in the microscope's
plane of focus. That helps out-of-focus areas stay dark, keeping a
specimen's fluorescence from being extinguished.
When Gao and Asano first tested their expanded mouse tissues on the
lattice scope, they saw a thicket of glowing nubs protruding from
neurons' branches. These nubs, called dendritic spines, often look like
mushrooms, with bulbous heads on skinny necks that can be hard to
measure. But the scientists were able to see even "the smallest necks
possible," Asano says, while simultaneously imaging synaptic proteins
nearby.
"It was incredibly impressive," says Betzig. The team was convinced
that they should explore the combined technique further. "And that's
what we've been doing ever since," he says.
The brain and beyond
Over the last two years, Gao and Asano have spent months at Janelia,
teaming up with biologists, microscopists, physicists, and computer
scientists across the campus to capture and analyze images. "This is
like an Avengers-level collaboration," Gao says, referring to the crew
of comic book superheroes.
Yoshinori Aso and the FlyLight team provided high-quality fly brain
specimens, which Gao and Asano expanded and used to collect some 50,000
cubes of data across each brain -- forming a kind of 3-D jigsaw puzzle.
Those images required complicated computational stitching to put the
pieces back together, work led by Stephan Saalfeld and Igor Pisarev.
"Stephen and Igor saved our bacon," Betzig says. "They dealt with all
the horrible little details of image processing and made it work on each
multi-terabyte data set."
Next, Srigokul Upadhyayula from Harvard Medical School, a co-first
author of the report, analyzed the combined 200 terabytes of data and
created the stunning movies that showcase the brain's intricacies in
vivid color. He and his coauthors investigated more than 1,500 dendritic
spines, imaged fatty sheaths that insulate mouse nerve cells,
highlighted all of the dopaminergic neurons, and counted all the
synapses across the entire fly brain.
The nuances of Boyden's team expansion technique make it well suited
for the lattice scope; the technique produces nearly transparent
samples. For the microscope, it's almost like looking through water,
rather than a turbid sea of molecular gunk. "The result is that we get
crystal clear images at blazingly fast speeds over very large volumes
compared to earlier microscopy techniques," Boyden says.
Still, challenges remain. As with any kind of super resolution
fluorescence microscopy, Betzig says, 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 artifacts to be introduced.
Because of this, he says, "we worked very hard to validate what we've
done, and others would be well advised to do the same."
Now, Gao and the Janelia team are 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," Boyden says.
Journal Reference:
- Ruixuan Gao, Shoh M. Asano, Srigokul Upadhyayula, Igor Pisarev, Daniel E. Milkie, Tsung-Li Liu, Ved Singh, Austin Graves, Grace H. Huynh, Yongxin Zhao, John Bogovic, Jennifer Colonell, Carolyn M. Ott, Christopher Zugates, Susan Tappan, Alfredo Rodriguez, Kishore R. Mosaliganti, Shu-Hsien Sheu, H. Amalia Pasolli, Song Pang, C. Shan Xu, Sean G. Megason, Harald Hess, Jennifer Lippincott-Schwartz, Adam Hantman, Gerald M. Rubin, Tom Kirchhausen, Stephan Saalfeld, Yoshinori Aso, Edward S. Boyden, and Eric Betzig. Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science, 2019 DOI: 10.1126/science.aau8302
Courtesy: ScienceDaily
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