Two-photon microscope provides unprecedented brain-imaging ability


Date:
December 2, 2021
Source:
University of California - Santa Barbara
Summary:
Advancing our understanding of the human brain will require new
insights into how neural circuitry works in mammals, including
laboratory mice.

These investigations require monitoring brain activity with a
microscope that provides resolution high enough to see individual
neurons and their neighbors.



FULL STORY ========================================================================== Advancing our understanding of the human brain will require new insights
into how neural circuitry works in mammals, including laboratory
mice. These investigations require monitoring brain activity with
a microscope that provides resolution high enough to see individual
neurons and their neighbors.


========================================================================== Two-photon fluorescence microscopy has significantly enhanced researchers' ability to do just that, and the lab of Spencer LaVere Smith, an associate professor in the Department of Electrical and Computer Engineering at UC
Santa Barbara, is a hotbed of research for advancing the technology. As principal investigator on the five-year, $9 million NSF-funded Next
Generation Multiphoton Neuroimaging Consortium (Nemonic) hub, which was
born of President Obama's BRAIN Initiative and is headquartered at UCSB,
Smith is working to "push the frontiers of multi-photon microscopy for neuroscience research." In the Nov. 17 issue of Nature Communications,
Smith and his co-authors report the development of a new microscope
they describe as "Dual Independent Enhanced Scan Engines for Large Field-of-view Two-Photon imaging (Diesel2p)." Their two- photon microscope provides unprecedented brain-imaging ability. The device has the largest
field of view (up to 25 square millimeters) of any such instrument,
allowing it to provide subcellular resolution of multiple areas of
the brain.

"We're optimizing for three things: resolution to see individual neurons,
a field of view to capture multiple brain regions simultaneously, and
imaging speed to capture changes in neuron activity during behavior,"
Smith explained.

"The events that we're interested in imaging last less than a second,
so we don't have time to move the microscope; we have to get everything
in one shot, while still making sure that the optics can focus ultrafast
pulses of laser light." The powerful lasers that drive two-photon imaging systems, each costing about $250,000, deliver ultrafast, ultra-intense
pulses of light, each of which is more than a billion times brighter
than sunlight, and lasts 0.0001 nanosecond.

A single beam, with 80 million pulses per second, is split into two wholly independent scan engine arms, enabling the microscope to scan two regions simultaneously, with each configured to different imaging parameters.

In previous iterations of the instrument, the two lasers were yoked
and configured to the same parameters, an arrangement that strongly
constrains sampling. Optimal scan parameters, such as frame rate and scan region size, vary across distributed neural circuitry and experimental requirements, and the new instrument allows for different scan parameters
to be used for both beams.

The new device, which incorporates several custom-designed and custom- manufactured elements, including the optical relays, the scan lens, the
tube lens and the objective lens, is already being broadly adopted for
its ability to provide high-speed imaging of neural activity in widely scattered brain regions.



========================================================================== Smith is committed to ensuring open access to the instrument. Long
before this new paper was published, he and his co-authors released
a preprint that included the engineering details needed to replicate
it. They also shared the technology with colleagues at Boston University,
where researchers in Jerry Chen's lab have already made modifications
to suit their own experiments.

"This is exciting," Smith said. "They didn't have to start from scratch
like we did. They could build off of our work. Jerry's paper was
published back-to-back with ours, and two companies, INSS and CoSys,
have sold systems based on our designs. Since there is no patent, and
won't be, this technology is free for all to use and modify however they
see fit." Two-photon microscopy is a specialized type of fluorescent microscopy. To perform such work in Smith's lab, researchers genetically engineer mice so that their neurons contain a fluorescent indicator
of neuron activity. The indicator was made by combining a fluorescent
protein from jellyfish and a calcium- binding protein that exists in
nature. The approach leverages the brief, orders-of-magnitude increase
in calcium that a neuron experiences when firing.

When the laser is pointed at the neuron, and the neuron is firing, calcium comes in, the protein finds the calcium and, ultimately, fluoresces.

Two-photon imaging enhances fluorescence microscopy by employing the
quantum behavior of photons in a way that prevents a considerable
amount of out-of- focus fluorescence light from being generated. In
normal optical microscopy, the light from the source used to excite the
sample enters it in a way that produces a vertical cone of light that
narrows down to the target focus area, and then an inverted cone below
that point. Any light that is not at the narrowest point is out of focus.

The light in a two-photon microscope behaves differently, creating a
single point of light (and no cones of light) that is in sharp focus, eliminating all out-of-focus light from reaching the imaging lens. "The
image reveals only light from that plane we're looking at, without much background signal from above or below the plane," Smith explained. "The
brain has optical properties and a texture like butter; it's full of
lipids and aqueous solutions that make it hard to see through. With normal optical imaging, you can see only the very top of the brain. Two-photon
imaging allows us to image deeper down and still attain sub-cellular resolution." Another advantage of two-photon excitation light is that
it uses lower-energy, longer-wavelength light (in the near-infrared
range). Such light scatters less when passing through tissue, so it can
be sharply focused deeper into tissue.

Moreover, the lower-energy light is less damaging to the sample than
shorter wavelengths, such as ultraviolet light.

Smith's lab tested the device in experiments on mice, observing their
brains while they performed tasks such as watching videos or navigating
virtual reality environments. Each mouse has received a glass implant in
its skull, providing a literal window for the microscope into its brain.

"I'm motivated by trying to understand the computational principles in
neural circuitry that let us do interesting things that we can't currently replicate in machines," he said. "We can build a machine to do a lot
of things better than we can. But for other things, we can't. We train teenagers to drive cars, but self-driving cars fail in a wide array of situations where humans do not.

The systems we use for deep learning are based on insights from the
brain, but they are only a few insights, and not the whole story. They
work pretty well, but are still fragile. By comparison, I can put a
mouse in a room where it has never been, and it will run to someplace
where I can't reach it. It won't run into any walls. It does this super reliably and runs on about a watt of power.

"There are interesting computational principles that we
cannot yet replicate in human-made machines that exist in
the brains of mice," Smith continued, "and I want to start
to uncover that. It's why I wanted to build this microscope." ========================================================================== Story Source: Materials provided by
University_of_California_-_Santa_Barbara. Original written by James
Badham. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Che-Hang Yu, Jeffrey N. Stirman, Yiyi Yu, Riichiro Hira, Spencer L.

Smith. Diesel2p mesoscope with dual independent scan engines for
flexible capture of dynamics in distributed neural circuitry. Nature
Communications, 2021; 12 (1) DOI: 10.1038/s41467-021-26736-4 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/12/211202123008.htm

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