The diffraction-image flow cytometer in ECU’s biomedical laser
laboratory would be conspicuously out of place in a hospital or in the
R&D department of some high-tech pharmaceutical company. Its
flickering lasers, twisting courses of plastic tubing, and gauges that
look ripped from a Cold War-era fighter jet are a far cry from the
bland plastic paneling of today’s commercial scientific equipment.
Rather, the device being developed here at ECU looks like what it is—a
real, honest-to-goodness science project. But for all of its aesthetic
quirks, the science behind it is nothing short of stunning.
Physics professor Dr Xin-Hua Hu and his revolutionary
diffraction-image flow cytometer. The device is used to study the
morphology of individual biological cells by collecting and analyzing
scattered laser light signals.
The brainchild of ECU physics professor Dr. Xin-Hua Hu, the
diffraction-image flow cytometer is the first device of its kind and is
a far-reaching step forward in increasing our understanding of cellular
A flow cytometer is a diagnostic tool used by researchers and medical
clinicians to study individual cells. It uses a laser and electronic
detectors called photomultipliers to measure particular characteristics
of cells as they are funneled through fast-moving liquid layers. The
flow cytometer gets its name from the pressure exerted on the core
liquid solution which forces it to stretch and narrow into a column the
thickness of one cell. This flow of single-file cells passes through
the laser beam allowing the electronic detectors to collect light data
on individual cells, and it allows them to collect that data at
incredible speeds. The fastest flow cytometers can easily process 1,000
cells a second.
Early research into eye applications for lasers introduced Hu into the
field of biomedical physics. Before joining the physics department at
ECU in 1995, he worked as a research scientist for a company that
manufactured biomedical lasers for use in cornea surgery. His expertise
in the field led to his participation in a five-year study of laser
tissue cutting, which prompted him to ask deeper questions about the
interaction of biological cells and the highly coherent light of a
“In order to understand how a laser beam cuts tissue, we first need to
know how the light from the laser is distributed through the tissue.
That is very difficult to determine because tissue severely scatters
light. So when we started to think about why tissue scatters light, we
started thinking about how cells scatter light, because tissue is made
up of cells. So that brought us to where we are now,” said Hu. “When I
started looking around for the best way to measure scattered light from
cells, I realized that a flow cytometer had the potential to rapidly
provide the scattered light signals from individual cells that I needed
to analyze large cell populations.”
However, for Hu to measure exactly how cells scatter light, he would
need to take the principle of the flow cytometer and significantly
improve upon its design. The problem with the current commercially
available flow cytometers is that they only collect data based on
fluorescence—one of the two results of highly coherent light striking
an object. The other result, scattered light, is essentially ignored.
The diffraction-image flow cytometer shines a laser beam at individual cells in a flow chamber.
Fluorescence is the luminescence resulting from the absorption of
light energy at one wavelength and the reradiation of that energy at a
different wavelength. Since normal cells are mostly transparent and
have no color for light to excite, a stain must be applied to a cell to
detect fluorescence. This means that researchers or clinicians must
target different molecules in the cells with stain. It can confirm the
existence of targeted molecules, but not where inside a cell these
Scattered light can provide researchers with information that
fluorescence cannot. By its very nature, scattered light contains far
more information about the three-dimensional structure of a cell due to
the high coherence of the man-made laser light. Much like how our brain
makes sense out of scattered noncoherent light from the sun or a light
bulb striking an object, new technology would be needed to make sense
out of the scattered coherent laser light.
Enter East Carolina University.
With collaboration from associate professor of physics Dr. Jun Quing
Lu, and ECU physics department staff member, part-time PhD student, and
hardware guru, Ken Jacobs, Hu set out build a new kind of flow
cytometer to record scattered coherent light as diffraction images.
“We already knew a lot about how cells scatter light, so I figured if
we could apply what we knew, we might be able to make a much better
instrument,” he said.
Hu’s vision was to create a flow cytometer that maintained the core
principles of traditional flow cytometer mechanics, while improving the
flow chamber and adding the ability to analyze cell morphology by
recording the diffraction image of the cell’s interaction with laser
light. He could then utilize that data to determine the
three-dimensional structure of cells via a sophisticated computer
program. But to do that, Hu first had to find a way to collect
His solution was to replace the light detectors of traditional flow
cytometers, with a fast charge-coupled device, or CCD, camera. CCDs are
commonly found in consumer digital cameras. They allow an image to be
converted into digital data for analysis. For his flow cytometer, Hu
uses an electronically cooled 16-bit CCD camera and microscope lens to
collect the vast data produced by scattered light.
“Each pixel of our camera is an electronic detector. So instead of
using one, or two, or 10 light detectors like existing flow cytometers,
we are using two million detectors because our camera has two million
pixels. It gives us much better knowledge of the scattered light
signal,” said Hu.
Jacobs constructed the device, which exists currently as a prototype in
ECU’s biomedical laser lab. In his more than 20 years in the physics
department, he has built or helped build many research devices and he
is excited by the potential of his latest project.
“No one understood where disease came from until they started looking
through a microscope,” he said. “Well, this is a different kind of
microscope. A regular microscope uses regular, everyday noncoherent
light. This uses laser light. It’s different light, so it gives you
different information. No one has found a good way to do this before
for analyzing 3D structures of cell.”
Much of the flow cytometer mechanics rely on hydraulic pressure to
produce the cell flow. The CCD camera is the blue square in the upper
right of this photograph.
While the hardware side of the diffraction image flow cytometer
presented some challenges—such as figuring out how to get the camera to
take an exposure at the precise moment a cell passed through the laser
beam with little background noise—much of the technology already
existed. But for her part in the project, Lu would need to develop and
write an entirely new computer program to make sense of all the data
the new flow cytometer could capture.
“What we are making here is actually two parts,” said Hu. “There is a
hardware component and a software component. Dr. Lu has been working on
the software, the computing algorithm we use for 3D modeling and
diffraction pattern analysis, for the past eight years. We have
received two grants from the National Institutes of Health to support
It is this software that truly sets the diffraction image flow
cytometer apart from what is currently available. Scientists have long
known the immense quantity of information stored in scattered coherent
light, but there was never a good way of acquiring and processing that
information with a flow cytometer. In other words, the data was simply
overlooked. And while this software is still early in development, it
will hopefully make this data not only relevant, but revolutionary. If
successful, it will give researchers a comprehensive knowledge of the
three-dimensional structure of an individual cell and all of its
component parts by reading the diffraction pattern of the scattered
Hu, Lu, and Jacobs are listed as inventors on a pending international
patent application by ECU on the equipment and methodology of the
diffraction-image flow cytometer, and are currently awaiting approval
of their invention. It is Hu’s second pending patent application while
at ECU, having already invented a new tissue imaging technique for
noninvasive diagnosis of lesions.
The 2007 commercial market for flow cytometers was more than $1.1
billion annually. They are used by researchers and by medical
clinicians for diagnosing diseases, especially blood diseases like
leukemia. Hu is very optimistic about the potential of his device and
its ability to help those in need.
“Right now, the blood analyzers in hospitals can only tell you what
cells are in the blood. That’s fine for a regular checkup, but what if
a patient has leukemia, which prohibits them from producing mature
white blood cells? The white blood cells are there, so the current
machines can detect them, but they aren’t mature cells so they don’t
work. You can’t use a traditional flow cytometer because they aren’t
good at explaining the structure of cells. Our device will have that
capability,” he said.
It will be years before the diffraction-image flow cytometer and its
software will be refined to the point of commercialization. But when it
is, it will be another success stories for ECU biomedical research.
Learn more about research at ECU and ECU’s Department of Physics.