Researchers Demonstrate Hyperspectral Imaging for Multiphoton Microscopy

WASHINGTON–(BUSINESS WIRE)– Traditionally, the study of diseases at the molecular level has required
scientists to extract cells and tissues from animal models and then look
for clues in the samples that can determine the mechanisms underlying
the disease and driving its progression. According to Chris B. Schaffer,
associate professor of biomedical engineering at Cornell
University, New York, USA, that is “like a person guessing who is
winning a battle based on a single photograph from the warzone.”

A better way, Schaffer said, is a method known as multiphoton imaging,
by which individual cells can be tracked in living tissue using
fluorescent labels and their three-dimensional structures visualized in
detail while functioning in a natural environment. However, current
multiphoton imaging systems can “become confused” when two or more
fluorophores (the fluorescent labels) are used together to examine the
behavior and interactions of different cell types. Called hyperspectral
multiphoton microscopy, a new innovation by Schaffer’s lab significantly
extends the current capability of this imaging technology by allowing
users to see and distinguish more colors of fluorophores at the same
time.

This new solution to the problem will be presented by the research team
at the Frontiers
in Optics (FiO) / Laser Science (LS) conference in Rochester, New
York, USA on 17-21 October 2016.

To use traditional multiphoton microscopy, cells of interest are tagged
with a fluorescent dye or genetically encoded fluorescent markers such
as green fluorescent protein (GFP) and then hit with a high-power,
short-pulse infrared wavelength laser. These fluorophores are excited —
and define the cells they tag — only at the laser focus, where the laser
energy is concentrated to create a high intensity.

“By scanning the laser focus throughout the sample, a 3-D reconstruction
of all labeled cells and tissues is generated,” Schaffer said. “These
images can be collected rapidly, giving researchers the ability to track
single cells in a living system, which is more relevant to understanding
human disease than tracking cells in a Petri dish.”

The problem with many fluorophores, Bares said, is that they aren’t one
distinct color.

“A red fluorophore, for example, will emit light across a range of
wavelengths, say from 600 to 650 nanometers, so that there’s overlap
with a red-orange fluorophore emitting light from 580 to 620
nanometers,” she explained. “The photodetector used to locate and
visualize the target cells has no way to tell which one is the source
for a 600-nanometer signal, and that’s bad, because the red and
red-orange fluorophores may mark cells with very different biological
functions.”

Bares said that in the Cornell hyperspectral technique, the entire
visible light spectrum is used to better characterize a sample.

“It’s similar to a simple multicolor imaging system, the standard
red-green-blue, or RGB, signal used in television where an image has
three color channels and each pixel gets a value in each of the three
channels,” she said. “In hyperspectral imaging, we collect the same
image over 48 color channels, yielding multiple values for each pixel —
and that provides a wealth of data for distinguishing very precisely
between fluorophores, and in turn, cells.”

To enhance its technique even further, the Cornell team used different
excitation laser colors. “For example, we eliminate the red versus
red-orange overlap problem completely if we collect images first using a
laser that only excites red fluorophores and then repeat the process
with another laser that only stimulates red-orange labels,” Bares said.

Schaffer, Bares and their colleagues have demonstrated the capabilities
of hyperspectral imaging with a 48-channel multiphoton microscope in a
number of samples, including identifying 10 different colors of
fluorescent beads in agarose, tracking proteins of interest in living
cell cultures by fusing them with fluorescent proteins, and
distinguishing between five cell types in the dense cortical tissues of
a live mouse brain. With more refinement — such as improving the image
analysis algorithms, increasing imaging speed and providing real-time
image processing — the researchers believe that their technique has a
very promising future.

“Having access to a microscope that allows researchers to see and
distinguish all of the cells in a tissue volume, as well as determine
which interactions between these cells cause disease symptoms, could
speed the development and deployment of new therapies,” Schaffer said.

About the Presentation

The presentation, “Hyperspectral Imaging in Live Mouse Cortex Using a
48-Channel Multiphoton Microscope,” by A.J. Bares, M.A. Pender, M.A.
Mejooli, S. Tilley, K.E. Chen, J. Dong, P.C. Doerschuk and C.B.
Schaffer, will take place from 15:45-16:00, Thursday, 20 October 2016,
in the Grand Ballroom D, Radisson Hotel Rochester Riverside, Rochester,
New York, USA.

Media Registration: A media room for credentialed press and
analysts will be located on-site in the Radisson Hotel. Media interested
in attending the event should register on the FiO website media center: Media
Center.

About FiO/LS

Frontiers in Optics (FiO) 2016 is The Optical Society’s (OSA) 100th
Annual Meeting and held together with Laser Science, the 32th annual
meeting of the American Physical Society (APS) Division of Laser Science
(DLS). The two meetings unite the OSA and APS communities for five days
of quality, cutting-edge presentations, in-demand invited speakers and a
variety of special events spanning a broad range of topics in optics and
photonics—the science of light—across the disciplines of physics,
biology and chemistry. The exhibit floor will feature leading optics
companies, technology products and programs. More information at: FrontiersinOptics.org.

About The Optical Society

Founded in 1916, The Optical Society (OSA) is the leading professional
organization for scientists, engineers, students and entrepreneurs 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: osa.org/100.

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