Opening new doors for biomedical and neuroscience research, Elizabeth
Hillman, associate professor of biomedical engineering at Columbia Engineering
and of radiology at Columbia University Medical Center (CUMC), has developed a
new microscope that can image living things in 3D at very high speeds.
In doing so, she has overcome some of the major hurdles faced by existing
technologies, delivering 10 to 100 times faster 3D imaging speeds than laser
scanning confocal, two-photon, and light-sheet microscopy. Hillman's new
approach uses a simple, single-objective imaging geometry that requires no
sample mounting or translation, making it possible to image freely moving
She calls the technique SCAPE, for swept confocally aligned planar
excitation microscopy. Her study is published in the Advance Online Publication
(AOP) on Nature Photonics's website on January 19, 2015.
"The ability to perform real-time 3D imaging at cellular resolution in
behaving organisms is a new frontier for biomedical and neuroscience research,"
says Hillman, who is also a member of Columbia's Mortimer B. Zuckerman Mind
Brain Behavior Institute. "With SCAPE, we can now image complex, living things,
such as neurons firing in the rodent brain, crawling fruit fly larvae, and
single cells in the zebrafish heart while the heart is actually beating
spontaneously—this has not been possible until now."
Highly aligned with the goals of President Obama's BRAIN Initiative, SCAPE
is a variation on light-sheet imaging, but, "It breaks all the rules," says
Hillman. While conventional light-sheet microscopes use two awkwardly
positioned objective lenses, Hillman realized that she could use a
single-objective lens, and then that she could sweep the light sheet to
generate 3D images without moving the objective or the sample.
"This combination makes SCAPE both fast and very simple to use, as well as
surprisingly inexpensive," she explains. "We think it will be transformative in
bringing the ability to capture high-speed 3D cellular activity to a wide range
of living samples."
SCAPE is an urgently needed breakthrough. The emergence of fluorescent
proteins and transgenic techniques over the past 20 years has transformed biomedical
research, even delivering neurons that flash as they fire in the living brain.
Yet imaging techniques that can capture these dizzying dynamic processes have
lagged behind. Although confocal and two-photon microscopy can image a single
plane within a living sample, acquiring enough of these layers to form a 3D
image at fast enough rates to capture events like neurons actually firing has
become a frustrating road-block.
While SCAPE cannot yet compete with the penetration depth of conventional
two-photon microscopy, Hillman and her collaborators have already used the
system to observe firing in 3D neuronal dendritic trees in superficial layers
of the mouse brain. In small organisms, including zebrafish larvae, SCAPE can
see through the entire organism.
By tracking these tiny, unrestrained creatures in 3D at high speeds, SCAPE
can capture both cellular structure and function and behavior. SCAPE can also
be combined with optogenetics and other tissue manipulations during imaging
because, unlike other systems, it does not require any movement of the imaging
objective lens or the sample to create a 3D image.
Hillman and her students built their first SCAPE system using inexpensive
off-the-shelf components. Her "aha" moment came when, looking at an old polygonal
mirror in the lab, she realized how it could be used to generate SCAPE's
unusual scanning geometry.
After several years of trial and error, Hillman and graduate student Matthew
Bouchard came up with a configuration that worked, and beautiful images started
to flow out. "It wasn't until we built it that we realized it was a light-sheet
microscope!" says Hillman. "It took us a while to realize how versatile the
imaging geometry was, how simple and inexpensive the layout was—and just how
many problems we had overcome."
Beyond neuroscience, Hillman sees many future applications of SCAPE
including imaging cellular replication, function, and motion in intact tissues,
3D cell cultures, and engineered tissue constructs, as well as imaging 3D
dynamics in microfluidics, and flow-cell cytometry systems—all applications
where molecular biology is delivering tools and techniques, but imaging methods
have struggled to keep up.
Hillman also plans to explore clinical applications of SCAPE such as
video-rate 3D microendoscopy and intrasurgical imaging. Next-generation
versions of SCAPE are in development that will deliver even better speed,
resolution, sensitivity, and penetration depth.
As a member of the new Zuckerman Institute and the Kavli Institute for Brain
Science at Columbia, Hillman is working with a wide range of collaborators,
including Randy Bruno (associate professor of neuroscience, Department of
Neuroscience), Richard Mann (Higgins Professor of Biochemistry and Molecular
Biophysics, Department of Biochemistry & Molecular Biophysics), Wesley
Grueber (associate professor of physiology and cellular biophysics and of
neuroscience, Department of Physiology & Cell Biophysics), and Kimara
Targoff (assistant professor of pediatrics, Department of Pediatrics), all of
whom are starting to use the SCAPE system in their research.
"Deciphering the functions of brain and mind demands improved methods for
visualizing, monitoring, and manipulating the activity of neural circuits in
natural settings," says Thomas M. Jessell, co-director of the Zuckerman
Institute and Claire Tow Professor of Motor Neuron Disorders, the Department of
Neuroscience and the Department of Biochemistry and Molecular Biophysics at
Columbia. "Hillman's sophistication in optical physics has led her to develop a
new imaging technique that permits large-scale detection of neuronal firing in
three-dimensional brain tissues. This methodological advance offers the
potential to unlock the secrets of brain activity in ways barely imaginable a
few years ago."
Hillman's technology is available for licensing from Columbia Technology
Ventures and has already attracted interest from multiple companies.
This research was supported by the following grants: NIH (NINDS)
R21NS053684, R01 NS076628 and R01NS063226, NSF CAREER 0954796, the Human
Frontier Science Program and the Wallace H. Coulter Foundation (E.M.C.H.), NIH
(NINDS) R01 NS069679 and the Dana Foundation (R.M.B.), (NINDS) R01NS070644
(R.S.M.), (NINDS) R01NS061908 (W.B.G.), DoD MURI W911NF-12-1-0594 (Yuste). M.B.
received NSF and NDSEG graduate fellowships. V.V. was funded by an NSF IGERT
Fellowship. C.S.M. is supported by a postdoctoral fellowship from Fundação para
a Ciência e a Tecnologia, Portugal.
A patent related to this technique issued on December 31st 2013 (inventors
Hillman and Bouchard). The authors are currently in licensing discussions.