Inspired by Combat Amputees, NRL is First to Image Cell Protein Secretions in Real-Time

Dr.Dr. Marc Raphael (center), with Joseph Christodoulides (left) and Dr. James Delehanty (right), invented a way to measure proteins secreted by a cell. We're developing a technique that is easily applicable for the biologists to gain a new window into what's happening in the problems they're already studying, says Raphael.
(Photo: U.S. Naval Research Laboratory/Jamie Hartman)

Dr. Marc Raphael, a physicist at the U.S. Naval Research Laboratory (NRL), leads a team that has become the first to image protein secretions from cells in real-time using live-cell microscopy. A major breakthrough for biology and medicine, they've invented a way to put protein-specific nanosensors on microscope coverslips; the sensors change in brightness as the cell sends proteins into its environment.

We're developing a technique that is easily applicable for the biologists to gain a new window into what's happening in the problems they're already studying, says Raphael. I think here's a place where nanotechnology could help.

Cells communicate with each other by secreting proteins; and by detecting secretions from other cells, which they accomplish by displaying highly specific receptors at the outer membrane, like open puzzle pieces. Raphael wondered, How can we monitor a cell's local environment, so we can essentially listen in on this signaling pathway?

The NRL team fabricated hundreds of gold nanosensors into a tiny, grid-like array. Using hybridoma cells, they demonstrated the sensors light up as they're exposed to secreted antibodies. But it's more subtle than just on and off: the sensor response varies according to the concentration of antibodies, brightening second-to-second and depending on the distance from the secreting cell.

ConcentrationNRL's grid-like array of gold nanosensors lets scientists measure antibodies secreted by a cell over time and space. The colors in this image show how the concentration of antibodies is highest closest to the cell.
(Photo: U.S. Naval Research Laboratory)

When a cell makes a decision, says Raphael, it's measuring the concentration of a chemical, it could be a drug or whatever, and determining its behavior from it. With a data analysis methodology created by colleague Jeff Byers, they quantified the concentration of antibodies over time. Says Raphael, And that's when you say, 'Okay, I've really got a window into what the cell is measuring.'

The team discovered something that, because the technology wasn't there, had never before been seen. These cells can secrete in bursts, says Raphael.

Biologists today can see proteins inside a cell by tagging them with fluorescent molecules. But in every image, says Raphael, the environment around the cell is displayed as black. Fluorescent tags either block the protein from crossing the outer membrane; or, if they do cross, their glow becomes too diffuse to be quantified. Raphael's invention can be used along with fluorescence to better illuminate what's happening in and outside the cell.

Nanosensors could explain why IED-related amputees have pain, complications

Raphael's admission, I'd never taken a biology course in college, may come as a surprise; his lab at NRL is actually in Materials and Sensors. But, he says, bone is a material, your skin is a material.

Cpl.Cpl. Anthony McDaniel, who was injured after stepping on an IED, competed in track and basketball in the 2012 Marine Corps Trials. NRL's Dr. Marc Raphael has devoted his career to amputees who suffer IED-related bone cell complications. We know it's a chemical communication, says Raphael. Cells are constantly monitoring their local environment, and altering that environment with their own secretions.
(Photo: Lance Cpl. Daniel Wetzel, USMC)

He credits his wife, Etaine, for turning his attention on this challenge in cell biology. For eight years, she worked with amputees at Walter Reed National Military Medical Center as a physical therapist. Part of our dinner conversation, Raphael says, would be about patients with leg amputations following injury from an improvised explosive device [IED]. In most IED-related amputations (possibly 64 percent), the bone doesn't seal off; rather, it pushes out painful growths, termed heterotopic ossification (HO).

You see this in blast injuries specifically, he says; it almost never happens after amputations from other causes, like motorcycle accidents or diabetes. The most common treatment is surgery to remove the excess bone. But, Raphael says, If it gets bad enough, additional amputations may be required.

Raphael was moved by these patients' struggles, and frustrated so little was known about what caused HO. What we started asking was, why do these [bone] cells want to grow?

So he started researching how these materials respond to injury. When you just cut your skin, Raphael says, there are layers of skin, there's pores, there's neurons, there's glands. And it all just gets wrecked. And somehow your body figures out how to put it all together again. And then you take a blast injury, which is immensely more complex compared to what we're talking about with a cut.

How do cells repair this complex architecture, and do it with perfect timing and with everything in its proper place? We know it's a chemical communication, says Raphael. Cells are constantly monitoring their local environment, and altering that environment with their own secretions.

A standard chip, hiding nano-power, reveals a bursting surprise

Just across the NRL campus from Raphael, Joseph Christodoulides fabricates the nanosensor arrays in a world-class cleanroom. The Nanoscience Institute [NSI] has just been so important for our research, says Raphael. That's what brought all this together. NSI gives funding and facilities access to novel, interdisciplinary NRL projects.

NanoplasmonicNRL has fabricated nanoplasmonic arrays to image cells protein secretions onto standard glass coverslips, reinforced with a silicon backing ring. The chip goes on a perfusion apparatus for fluid exchange (left), with similar dimensions as commercial petri dishes (right) to ensure compatibiliity.
(Photo: U.S. Naval Research Laboratory/Jamie Hartman)

Christodoulides integrates the arrays onto one-inch glass cover slips, which are standard in cell microscopy. You can't see the sensors on there [without a microscope] because they're so small, says Raphael. But a biologist would look at this and say, 'Oh yeah, it's the same kind of chip I use all the time.'

The chip fits into a live cell incubation assembly. The assembly includes fluidic tubes, to provide nutrients to the cell, and electrical contacts. The whole device is enclosed in an incubator attached to a light microscope, also standard for live cell microscopy.

So far, Raphael's team has only tested the nanosensor array with one type of cell as a proof of concept: hybridoma cells cultured by NRL biologist James Delehanty and picked because we knew they secreted antibodies at a healthy rate. But they've already discovered the cells don't just secrete steadily over time. Rather, they've seen the nanosensors occasionally get really bright over the course of a minute or two, indicating burst-like secretions.

Today, using commercially available instruments, a biologist can run an experiment to determine what a cell secreted over the last few hours or days. But we can update this on the order of seconds, says Raphael, while monitoring the cell visually, and tell you how its secretions are changing.

What the bursts signify—necrosis or some other process—is unknown, because until Raphael's invention there was no way to measure a burst like this.

NRL's interdisciplinary culture expands capabilities, applications

Dr.Dr. Marc Raphael of NRL looks at real-time images of a nanoplasmonic array brightening in response to a cell that's secreting proteins. He hopes his invention will help combat amputees. Interdisciplinary work is, in my opinion, it's the future of biology, he says. This is something where NRL can really make a difference.
(Photo: U.S. Naval Research Laboratory/Jamie Hartman)

Having proved the sensor works with a hybridoma, Raphael's team is now looking at protein growth factors associated with HO and many types of cancer. There is an obvious overlap [between HO and cancer], he says. Both problems are associated with cell division gone out of control.

Further along, he hopes to get to measuring secretions from bone cells, possibly collaborating with medical researchers to compare normal cells with cells from IED-related amputees.

Additionally, he is working with biologists Delehanty, George Anderson, and Jinny Liu to enhance the sensors. Instead of having a chip with every array targeted to the same proteins, they're looking to target multiple ones on the same chip. You could call it lab on a chip, he says, because we're working on a technique that will allow us to do multiplexing.

He's also collaborating with Dr. Marc Christophersen in the Space Science Division on making a new array that would lift the gold nanosensors up onto glass tips, like a surface of stalagmites. So this is one of our new ideas, says Raphael, we actually put the sensors on the ends of these pillars, so that we can probe just inside the cell membrane.

NRL’sDr. Marc Raphael of NRL places the nanotechnology his team invented to image protein secretions on a Zeiss Axio Observer microscope. They are working on enhancing the sensor with multiplexing and new fabrication techniques that would allow cell biologists to probe just inside a cell's membrane.
(Photo: U.S. Naval Research Laboratory/Jamie Hartman)

There are important signals that get transmitted at the cell membrane, which are difficult to measure with fluorescence. This is a major challenge for cell biology because, as Raphael says, So much happens at the cell membrane—at the interface between outside and inside. This is where [the cell] begins processing the information it's gathering from its local environment.

There are few places in the world where biologists, physicists, and space scientists can come together; fewer still also have the facilities to take inventions from concept to fabrication to testing all in one place. Interdisciplinary work is, in my opinion, it's the future of biology, says Raphael. This is something where NRL can really make a difference.

His wife's stories of amputees—who, told they won't walk again, run the Army 10 miler; who rock climb; who continue to serve—still drive his research. It's really inspiring, Raphael says. I don't know how far away we are, but even if I spend my career on this and I fail, I'd rather have tried.

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