ready for their close-up

Hollywood celebrities relish their photo ops, and exert a great deal of effort in putting their best face forward. But while biological entities like cancer cells and protein molecules aren't camera shy, they're not exactly photogenic either. It's tough to image such things in sufficient detail for meaningful scientific analysis, because they're so darn tiny to begin with. Fortunately for any spotlight-hungry biological molecules out there yearning for their 15 minutes of fame, physicists are constantly developing new and improved methods to get better pictures of these tiny objects that are so critical to human health. Even in biophysics, image counts, you see.

It helps to have a bit of sparkle, and it turns out that healthy (from the disease's perspective) cancer cells do shimmer impressively for the cameras -- CCD cameras, specifically -- with a new technique called digital holographic imaging that 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. (For tons of great links and handy tips on creating your own holograms, go here.)

Back in 2002, Nolte's group was the first to use holography to image the internal structure of tissue. Conventional microscopy techniques use 2D slides and therefore don't delve very deeply into tissue, which is essentially a 3D construct; 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. (Yes, it's not much, but that should give you an idea of how little conventional microscopy penetrates the surface of tissue.) Now he's combined holographic imaging with laser ranging -- a technique similar to radar, in that it 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 in a recent press release.

Nolte had more to say about his work in person at the APS March Meeting in Denver (yes, we are still here, but not for long!). He began by provocatively asking, "What is life," and concluded that life is basically motion. One might quibble philosophically (or atomically-physically) with that statement, if one were so inclined, but we are quite happy to accept the premise at face value for the purposes of this post. Besides, his communication instincts were spot on: it's a perfect opening for non-scientists, since we can look at a rock, and look at a bird, and determine that, indeed, the animate object is clearly in motion.

Nolte brought it up for a reason: his new imaging technique essentially 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, and therefore it's a primary target of many anti-cancer drugs, like colchicine, a derivative of the autumn crocus (a.k.a., "meadow saffron") traditionally used to treat gout. Colchicine does this by blocking the formation of the microtubules that make it possible for organelles to navigate around the cell. Microtubules are "the highways of the internal cellular structure," says Nolte.

His digital holographic imaging system creates a hologram of a tumor ( in this case, an osteogenic sarcoma -- bone cancer -- tumor in a rat), whose center is usually filled with dead (necrotic) tissue surrounded by an outer shell where the cells madly multiplying 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 (among other properties) 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, according to Nolte.) 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 displayed 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 of the drug and the time it takes each does to become significantly affected."

It's exciting stuff, plus the shimmery pictures are quite pretty. But Nolte's isn't the only research group finding innovative new ways to image biological molecules, thereby gleaning useful new knowledge about how they function.  Andre Brown of the University of Pennsylvania (who also blogs at Biocurious) has employed the force sensing mode (as opposed to the imaging 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. Fibrin forms around the damaged area in a mesh-like pattern, which dries and hardens to stop the bleeding -- a good thing for wound healing, when you want the blood to clot, and not so good if said blood clot results in a heart attack or stroke.

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 than other biological fibers before breaking -- including collagen, spider silk (known to be pretty darned rugged) 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.

So Brown saw an intriguing correlation between, say, 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 molecular level. And 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. (Brown wrote a very nice blog post on his recent work a few weeks ago, for those interested in a more technical description of the research. And for some nifty video footage of a fiber  in the process of stretching, go here.)

Today's post would not be complete without taking a moment to wish a very happy 50th birthday to the incomparable PZ Myers -- mild-mannered Midwestern biology professor by day, fire-breathing atheist who battles Creationist wingnuts by night. But what to get for the heathen biologist who has everything? Jen-Luc Piquant scoured the Internets for this nifty recipe for stir-fried squid in shrimp sauce -- complete with tantalizing how-to photos -- but it turns out that PZ craves poetry. We're much too lazy to emulate Richard Dawkins, who wrote PZ a birthday poem. (Harumphs Jen-Luc: "Show-off.")  Instead, we humbly offer this 17th century haiku by Matsuo Basho, who died in 1694 at the age of... 50. So it's especially a propos, especially if read over a steaming plate of stir-fried squid. Enjoy!

The Squid Seller's Call

The squid seller's call

mingles with the voice

of the cuckoo.

 

 

Comments

[partha m]

you had me until the squid....now I'm kinda revolted....where did I put that box of thin mints????

[Andre]

Thanks mentioning our work. I thought I recognized you, but I wasn't really sure since I only had your blog picture to go on. It was a pleasure to sort of meet you! I also sort of met Sean once. I like crossovers between the blogosphere and "real" world, but I imagine you both must love it!

[apalazzo]

Re cancer and colchicine: what Nolte describes is incorrect (if what you refer to as "organelles" are mitochondria and the like). When microtubule based drugs are used to nuke cancer cells, the drugs are used at very low concentrations to prevent microtubule growth and shrinkage. The decreased microtubule dynamics impacts the mitotic spindle and inhibits the attachment of chromosomes to the microtubules and the pulling of sister chromatids apart. As a result any cell that tries to divide can't do it and dies. So yes, if you add a ton of colchicine to cells all the microtubules will disappear and organelles such as mitochondria can't move around, but when the compound is used in patients there will be no impact on the movement of organelles. Now of course colchicine treatment causes dividing cells to die, and the organelles of dead cells don't move, so indirectly colchicine will impact cellular motion, but only by killing the rapidly dividing cells.

[uure]

Yes! so hold.a

[Jeramy]

Hi Jennifer.Thanks for your post about cancer.Your article make great idea to me.Sorry for my english.Thanks.