Biomolecules Ready for Their Close-Up with New Imaging Methods

Physicists are constantly developing new and improved methods to get better pictures of cancer cells and protein molecules that are so critical to human health. Two such methods were described by speakers at the APS March Meeting in Denver.

Cancer cells, for instance, shimmer impressively for CCD cameras when imaged with a new technique called digital holographic imaging. It produces time-lapsed, dynamic speckled images that “shimmer” in response to cellular motion. Recent work at Purdue University marks the first time holography has been used to study the effects of a drug on living tissue, according to David Nolte, the Purdue University physics professor who headed up the research.

Conventional microscopy techniques don’t delve very deeply into tissue. Nolte wanted to get a peek inside the tissue itself, preferably at a depth of about 1 millimeter, to gain a better understanding of its structure.

He has combined holographic imaging with laser ranging, which measures how long it takes for a laser pulse to travel to an object and be reflected back. “The holography gives us the peaks and valleys and detailed depth information, while the laser ranging allows us to control how deep we are looking,” he said.

Nolte’s new imaging technique measures the motion of organelles inside cancer cells to determine whether they’re living or dead. Organelles play a key role in fostering the out-of-control cancer cell division that so often proves fatal to the patient.

His imaging system creates a hologram of a tumor whose center is usually filled with necrotic tissue surrounded by an outer shell where the cells madly multiply with wild abandon. Laser light shines on both the object and the CCD camera, and the reflected light is fed into the system, which records very detailed information about depth and motion of the components at work in the tumor tissue.

All that outer shell activity shows up as a bright shimmer in the resulting image, while the dead tissue at the center doesn’t move at all (What little shimmer there is at the center can be attributed to the incidental motion of the CCD cameras recording the experiment). Using this technique, it’s possible to create handily color-coded “motility maps” of cellular activity at three different tissue depths: 120, 190, and 330 microns. Red indicates high activity, and is found at 120 microns. By 330 microns, that activity has slowed sufficiently that the contrast color is predominantly yellow. Completely dead tissue shows up as blue.

So cellular motion becomes a built-in contrast agent used to enhance the image, making digital holographic imaging a vital emerging tool in measuring the effectiveness of anti-cancer drugs like colchicine. If the drug is working, there will be a reduction in the motion of the organelles, which will show up with less shimmer in the image on the computer display, and can then be quantitatively analyzed.

“We have moved beyond achieving a 3D image to using that image for a direct physiological measure of what the drug is doing inside cancer cells,” said Nolte. “This provides valuable information about the effects of various doses of the drug and the time it takes each dose to become significantly affected.”

Nolte’s isn’t the only research group finding innovative new ways to image biological molecules. Andre Brown of the University of Pennsylvania has employed the force sensing mode of standard atomic force microscopy to image molecules of fibrin, a protein that acts as a molecular spring to keep blood clots structurally stable, but still flexible enough to allow blood to flow through them.

Fibrin develops in the blood from another protein called fibrinogen, when blood cells release the enzyme thrombin in response to encountering damaged tissue.

Last year, researchers at Wake Forest University, Harvard, and the University of North Carolina used AFM to test the stretchiness of fibrin, and found these fibers can stretch much further before breaking than other biological fibers–including collagen, spider silk, and keratin. That property is crucial to fibrin’s ability to stop the flow of blood, which exerts a great deal of mechanical stress on the fibers. There have also been studies demonstrating that fibrinogen taken from patients with heart problems forms stiffer clots than that taken from healthy control patients.

Brown saw an intriguing correlation between heart disease and the mechanics of fibrin in blood clotting, and thought that protein unfolding might play a role in the unusual elasticity (“stretchiness”) of fibrin fibers. He used AFM in combination with total internal reflection fluorescence microscopy to measure the force with which this unfolding occurs–marking the first time the mechanics of fibrinogen has been measured at the single molecule level.

He found that protein unfolding does indeed seem to play a role in the mechanics behind blood clotting. Next on the agenda is to explore whether this unfolding plays any kind of role in clot mechanics at more modest extensions.