(Image caption:
Using a new microscopy technique researchers simultaneously recorded
from the cortex (green) and hippocampus (blue) of a mouse brain. Bright
areas correlate with cell activity)

New microscopy technique peers deep into the brain

In order to understand the brain, scientists must be able to see the
brain—cell by cell, and moment by moment. However, because brains
comprise billions of microscopic moving parts, faithfully recording
their activity comes with many challenges. In dense mammalian brains,
for example, it is difficult to track rapid cellular changes across
multiple brain structures—particularly when those structures are located
deep within the brain.

A novel microscopy technique, developed by Rockefeller scientists,
integrates new and existing approaches to help build a more cohesive
picture of the brain. Described in Cell, the technology captures cellular activity across large volumes of neural tissue, with impressive speed and at new depths.

Laser focused

For decades, brain imaging has been plagued by trade-offs. Some
techniques produce beautiful images but fail to record neural activity
in real time. Others can keep up with the brain’s speed but have poor
spatial resolution. And although there are tactics that successfully
combine rapidity and image quality, they typically capture only a small
number of cells.

“This is in part because the limits that govern these tradeoffs have
not been explored or pushed in a systematic and integrated manner,” says
Alipasha Vaziri, head of the Laboratory of Neurotechnology and Biophysics.

Hoping to end the era of trade-offs, Vaziri recently endeavored to
improve upon a technique known as two-photon (2p) microscopy. It
involves the application of a laser that causes bits of brain tissue to
fluoresce, or light up; and for many researchers, 2p has long been the
gold standard for probing cellular activity in the brain.

Yet, this technique has limitations. Standard 2p microscopy requires
point-by-point scanning of a given region, which results in slow
imaging. To resolve this issue, Vaziri and his colleagues implemented a
novel strategy that permits recording from multiple brain regions in
parallel, while carefully controlling the size and shape of each spot

Another weakness of traditional 2p is that it measures only the
surface, or cortex, of the brain, neglecting structures buried deep
within the organ, such as the hippocampus, which is involved in storing

“One of the biggest challenges in neuroscience is developing imaging
techniques that measure the activity of deep brain regions while
maintaining high resolution,” says Vaziri.

Taking up this challenge, he decided to make use of a newer technology: three-photon
(3p) microscopy. Whereas 2P doesn’t reach beyond the surface, or
cortex, of a mouse brain, 3p penetrates deeper regions. Called hybrid
multiplexed sculpted light microscopy, or HyMS, Vaziri’s latest
innovation applies 2P and 3P concurrently, allowing researchers to
generate a picture of rapid cellular activity across multiple layers of
brain tissue.

Deep dive

In addition to its hybrid laser strategy, HyMS also integrates other
recent technical and conceptual advancements in the field—a synergistic
approach that, Vaziri says, guided the development of the technology.
The goal, he says, was to maximize the amount of biological information
that could be obtained through multi-photon excitation microscopy while
minimizing the heat produced by this method. And when testing their new
system, the scientists certainly obtained a lot of information.

HyMS boasts the highest frame rate of available 3p techniques, which
means it can capture biological changes at record speed. And whereas
previous techniques scanned only a single plane of tissue, this
technology can obtain information from the entire tissue sample and
allows users to record from as many as 12,000 neurons at once. Another
advantage of HyMS is its ability to simultaneously measure activity from
brain areas at different depths. Since different layers of the brain
constantly exchange signals, says Vaziri, tracking the interplay between
these regions is key to understanding how the organ functions.

“Before, people hadn’t even been able to look at the activity of
neurons over the entire depth of the cortex, which has multiple layers,
all at the same time,” he says. “With this technology you can actually
see what the information flow looks like within the cortex, and between
cortical and subcortical structures.”

In addition to probing new depths, HyMS allows researchers to record
brain activity from animals as they actively engage with their
environment. In a recent experiment, for example, the researchers used
the technology to record signals from thousands of mouse neurons as an
animal walked on a treadmill or listened to sounds. The fact that they
were able to obtain good recordings suggests that the technique may be
used to monitor large cell populations as animals perform diverse
tasks—an application that could help elucidate neural mechanisms
underlying various aspects of behavior and cognition.

Further, says Vaziri, techniques like HyMS will be vital to
researchers hoping to better understand how brains process information.
Neurons in the brain are densely interconnected and information is often
represented not by individual cells, but by states of the network.

“To understand the dynamics of a network,” he says, “you need to get
accurate measurements of big portions of the brain at a single-neuron
level. That’s what we’ve done here.”

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