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Enhanced 3D Imaging Poised to Advance Treatments for Brain Diseases


Microscope add-on tackles trade-offs in high-resolution imaging of
large tissue volumes

Researchers have developed a combination of commercially available
hardware and open-source software, known as PySight, that improves rapid
2D and 3D imaging of the brain and other tissues. By seamlessly enabling
integration of the fastest 3D imaging solution available today, the
advance in microscopy could help scientists to better understand brain
dynamics and discover new treatments for health problems such as stroke,
epilepsy and dementia.

In Optica,
The Optical Society's journal for high impact research, the researchers
describe PySight, which serves as a photon counting add-on for laser
scanning microscopes. Geared with this novel combination of software and
hardware, they improved the quality of 2D and 3D imaging of neuronal
activity in the living brain.

Because it can image deep into tissue, a laser-based technique known as
multiphoton microscopy is often used to study the rapid activity
patterns of neurons, blood vessels and other cells at high resolution
over time. This microscopy method uses laser pulses to excite
fluorescent probes, eliciting the emission of photons, some of which are
detected and used to form 2D and 3D images.

Trying to capture the full breadth of neuronal activity with multiphoton
microscopy forces scientists to image faster. As a result, fewer and
fewer photons become available to form images, much like taking a photo
with shorter and shorter exposure times. The challenge becomes how to
get meaningful images under these dim conditions.

"To overcome this hurdle, microscopists have used a detector-readout
method called photon counting," said research team leader Pablo Blinder
from Tel
Aviv University
in Israel. "However, because its implementation
required extensive electronics knowledge and custom components, photon
counting has never been widely adopted. In addition, commercially
available solutions were ill-suited to perform very fast imaging such as
required for 3D imaging."

In addition to advancing neural imaging research, PySight's improved
sensitivity could facilitate rapid intraoperative identification of
malignant cells in human patients using multiphoton microscopy.
PySight's novel approach for reconstructing 3D scenes could also improve
performance of light detection and ranging, or LIDAR. This could help
lower the costs of self-driving cars that use LIDAR to map their

Detecting single photons in 3D

PySight provides high spatiotemporal resolution while producing a data
stream that scales with the number of detected photons, not the volume
or area being imaged. "Conventional data acquisition hardware stores the
brightness of every pixel or 3D voxel even when it is zero because no
photons were detected in that particular location," Blinder explained.
"PySight, however, only stores the precise detection time of each
photon, allowing researchers to conduct rapid imaging of large volumes
over long sessions, without compromising spatial or temporal resolution."

To reconstruct a multidimensional image, knowing when each photon hits
the detector isn't enough. It's necessary to also know where it
originated in the brain. "This is even trickier if you want to simplify
the system and avoid synchronizing the different scanning elements,"
said Blinder. "To accomplish this, our software reads a list of photon
arrival times along timing signals from the scanning elements,
determines the origin of each photon within the sample and generates 3D
movies that can span three or more dimensions."

The photon arrival times are generated by a device known as a
multiple-event time digitizer, or multiscaler, which records the times
with a precision of 100 picoseconds. Another key component was an
off-the-shelf resonant axial scanning lens that changes the focal plane
hundreds of thousands of times per second. This lens was used to rapidly
scan the laser beam across different depths within the brain and allowed
the team to reconstruct continuous 3D images.

Easier, cost-effective, continuous 3D imaging

"The multiscaler we used hasn't been applied to neuroimaging because the
output isn't easy to interpret, and using a resonant axial scanning lens
for bioimaging has required custom-made hardware to generate 3D
renderings from the acquired data," said Blinder. "PySight turns the
output from both components into a 3D movie effortlessly. Because the
software is freely available to the public, it should greatly aid labs
previously deterred by the high technical barrier that accompanied 3D
imaging." Because of its generic application interface, PySight could
also be used to interpret similar photon detection time lists from
suitable hardware devices.

To test whether PySight was truly plug and play, the researchers walked
with their multiscaler to another imaging lab on the Tel Aviv University
campus. They were able to simply plug in the device, download the
PySight software and start recording odor responses in fruit flies
genetically modified to express voltage indicators — a fast probe for
neuronal activity — in a subset of their neurons.

In addition to continuing to improve the PySight software, the
researchers would like to add support for other microscopy imaging
methods such as fluorescence lifetime imaging, which relies on the
timing of each photon relative to its originating laser pulse. Because
the software is open source and provides direct access to photon arrival
times, it enables other scientists to add new features and meet their
specific needs.

Paper:H. Har-Gal, L. Golgher, S. Israel, D. Kain, O. Cheshnovsky,
M. Parnas, P. Blinder, "PySight: plug and play photon counting for fast
continuous volumetric intravital microscopy," Optica, 5, 9,
1104-1112 (2018). DOI:

About Optica

Optica is an open-access, online-only journal dedicated to the
rapid dissemination of high-impact peer-reviewed research across the
entire spectrum of optics and photonics. Published monthly by The
Optical Society (OSA), Optica provides a forum for pioneering
research to be swiftly accessed by the international community, whether
that research is theoretical or experimental, fundamental or applied. Optica
maintains a distinguished editorial board of more than 50 associate
editors from around the world and is overseen by Editor-in-Chief Alex
Gaeta, Columbia University, USA. For more information, visit Optica.

About The Optical Society

Founded in 1916, The Optical Society (OSA) is the leading professional
organization for scientists, engineers, students and business leaders
who fuel discoveries, shape real-life applications and accelerate
achievements in the science of light. Through world-renowned
publications, meetings and membership initiatives, OSA provides quality
research, inspired interactions and dedicated resources for its
extensive global network of optics and photonics experts. For more
information, visit

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