University Times

A CIM image showing a section of mouse small intestine in which toll-like receptor 4 is stained green, nucleus blue and filamentous actin red.

A confocal immunofluorescence microscopy (CIM) image showing cell death in hepatocytes (liver cells) from a green fluorescent protein (GFP) transgenic rat. The nuclei are stained orange, peroxisomes red and mitochondria blue. The cell at left has leaked its cytoplasmic GFP during the dying process but the intact, live hepatocyte at right retains its cytoplasmic GFP and remains green.

“When you go to any poster day at the medical school or anywhere, you’ll see our pictures everywhere,” says Simon C. Watkins, director of Pitt’s Center for Biologic Imaging.

CBI provides investigators with a wide range of cellular imaging techniques that can focus their view down to a single molecule. With more than two dozen microscopes and a staff of about 20 that includes four faculty members, it is among the largest, if not the largest such facility in the country, Watkins said. The center’s expertise in light and electron microscopy provides a glimpse into the tiniest of worlds, yielding big benefits for researchers.

Visitors to CBI’s suite in the Starzl Biomedical Science Tower need not look far for examples. One wall is a veritable library of journal covers featuring CBI microscopy images; another has row upon row of recently published articles to demonstrate CBI’s capabilities to prospective collaborators.

To lighten the atmosphere, poster-sized reproductions of famous works of art including Andy Warhol’s self-portrait, Grant Wood’s “American Gothic,” Van Gogh’s “Starry Night” and Edvard Munch’s “The Scream” up close reveal themselves to be photomosaics made up of tiny tiled images of tiny things.

The images are fascinating not only for their scientific value but for their intricacy and beauty as well. Electron microscopes yield black and white images; colors are added or contrast enhanced for clarity as photos are edited, said associate director Donna Stolz, who clearly enjoys both the scientific and the aesthetic value of the images.

A reconstruction of a 20-slice confocal stack of a zebrafish embryo in which the vasculature of the fish is seen in green.

For some people, simply having the data provided by the images is sufficient. “I’m more picky,” she said, admitting that she enjoys working on the photos after hours just for fun.

In addition to her scientific duties, Stolz is the creative force behind the Science as Art component of the University’s annual science symposium. The CBI lounge is strewn with science-themed pillows created for Science 2010. (See Oct. 14 University Times.) The photomosaics were created for Science 2009. The periodic table of electron microscopy compiled for Science 2008 includes “elements” such as an image of an ant (for antimony) taken via scanning electron microscopy and (for copper) a high-resolution view of the Abraham Lincoln statue as seen in the image of the Lincoln Memorial on the back of a penny. Stolz already is contemplating a video presentation for next year.

A SEM image of E. coli (green) being engulfed by a macrophage in a process known as phagocytosis.

“I find there are two different kinds of people here,” she said. “People who just take pictures to get the data and ‘pictures people’ who take a lot of time to take pictures that are more artistic. Nobody’s wrong or right, it’s just that everybody has a different way of looking at it.”

CBI’s images grace journal covers, enhance research publications and make colorful eye-catching works of art, but their real beauty lies beneath the surface — just a few millimeters deep.

The center houses some $10 million worth of equipment — some of which has come through grants, with other instruments on loan or placed for testing by industry. “When we have instrumentation that we get grants for, we’re generally working at the edge of what’s possible so we’re getting things that don’t exist elsewhere or we’re doing something novel,” Watkins said. “We have all the technologies that are available. The reason we’re successful is because we’re at the edge of what you can do.”

What sets Pitt’s CBI apart is the faculty component, he said. Other centers mostly are run by technicians. “They’re not driven by academics who really have a focused interest in the application of technology,” he said.

The faculty expertise is a crucial part of putting the equipment to the best scientific use. Watkins, who was recruited from Harvard Medical School’s Dana-Farber Cancer Institute to found Pitt’s center in 1991, is a tenured professor in the Department of Cell Biology and Physiology as well as in the Department of Immunology. Stolz also holds a faculty appointment in cell biology and physiology.

Collaboration is a key aspect of CBI. Its faculty members’ research expertise comes into play when other scientists seek to use the center. Watkins is the initial contact when new users want to begin a project involving CBI resources. Strategies are devised in conversations between CBI staff and the PI or his or her student.

A scanning electron micrograph (SEM) of podocyte pedicels (foot processes) in a mouse kidney. One podocyte is colored blue, the other yellow. This image is part of the playful periodic table of electron microscopy CBI compiled for Pitt’s Science 2008 symposium. To see the entire table, visit www.cbi.pitt.edu/gallery/Elements.

Often the imaging technique that researchers seek to use is based on images they’ve seen elsewhere. Many times, after discussing the proposed work, CBI staff are able to use their combined understanding of research and experience in microscopy techniques to suggest better alternatives.

“People come in here and they have an idea and they have no idea how to test it,” Watkins said. “They have this one figure from a paper. And because we have all these new ways of testing, we generally take them in a totally different direction.”

Watkins estimated that CBI faculty work with about 150 research groups per year and publish or collaborate on approximately 40 papers annually. He and Stolz each are involved in about 18 published papers per year, Watkins said.

Some collaborations have resulted in seminal work, such as the discovery that dendritic cells communicate with other distant cells by way of tunneling nanotubules, or TNTs. The new form of communication between cells was reported in papers published by Watkins and immunology professor Russell Salter in 2005.

Watkins noted that the publications on which CBI staff collaborate represent just a fraction of the research that is furthered through the center. Many scientists who use the microscopes publish the resulting research on their own.

Although some equipment is restricted, researchers can schedule scope time on many of the center’s instruments. During busy periods, some are booked 18-20 hours a day with researchers putting their names on a waiting list for time on the more popular ones.

On any given day, CBI’s microscopy suites may be filled with researchers studying problems in any number of scientific fields. While the main focus is biology and life sciences, “We work in collaboration in all areas,” Watkins said.

Pitt researchers make up the bulk of the clientele, but CBI also works with visiting researchers who don’t have access to such equipment on their home campus and offers services to biotech companies and other businesses on an hourly basis.

Microscopy appeals to Watkins’s innate problem-solving method. “I had this way I looked at the world, trying to solve problems by pulling them apart. The imaging technologies allow you to do that. You can put in indicator molecules — beacons that tell you where things are and tell you how much there is. We can look at things in living systems so we can see where things are going and how fast they’re going. We can do it at every level of resolution, from just a cluster of a few molecules to an entire animal.”

Traditionally, science has observed slices frozen in time, but advances in microscopy have made it possible to watch on a molecular level processes in living or nonliving material as they occur in their natural environment. “It gives a very rich and rounded image of what’s going on inside,” Watkins noted.

As a field, imaging is moving rapidly on many fronts. Improved camera technologies are coming to the market and better reagents — in particular the fluorescent proteins that enable cell processes to be observed — are being developed, Watkins said. “We have molecular beacons to tell us if something’s there or not, or how much of it is there,” he said.

New reagents can change color depending on their environment and can be turned on and off — advances that Watkins likened to the difference between having a single screwdriver in the toolbox to owning the entire Snap-On Tools catalog. “We’re getting more and more colors, more things, more rapidly,” Watkins said.

The future “is all about speed, imaging faster,” he said.

“Science generally likes to make things bigger so you can see things you couldn’t see with the naked eye or with some other method of measuring,” he noted. Cameras and microscopes alone often aren’t enough to create meaningful results. Heavy computing power is needed to handle data that not long ago would have been too unwieldy to manage. Watkins’s desktop computer has eight processors, 48 gigabytes of RAM and 24 terabytes of hard drive space. “If you didn’t have bigger and faster computers, you couldn’t deal with the analysis,” he said.

A single slice immunofluorescence confocal image of HeLa cells. Mitochondria are stained blue, peroxisomes green, filamentous actin red and nuclei orange.

Watkins’s current research involves imaging blood flow in baby zebrafish, work that only recently became possible. “I only image for a second or two, but I collect 1,000 pictures in one second, watching the individual blood cells move down the vessels. The heart will beat 4-5 times in that one second, so I can see the whole beat,” he explained. During the high-speed imaging, he estimated his microscope generates 100 megabytes of data per minute.

Two seconds’ worth of fish blood flow data could take three hours to analyze, he said, noting that 23,000-30,000 blood cells may travel down the blood vessel in that short period of time.

“We have a continual need for bigger, faster, better computers,” he said. “Without the computers and the analysis we’d have nothing.”

To see more examples of CBI’s work, go to www.cbi.pitt.edu.

—Kimberly K. Barlow