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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 biology.

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 laser beam.

“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.

Laser shines through cell flow

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 molecules reside.

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 scattered light.

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 this research.”

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 light signal.

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.


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