On a sunny, cold, mid-February morning, Simon Watkins and Claudette St Croix are getting familiar with the insides of a zebra fish embryo. The translucent organism, about one-eighth the size of a kid’s thumbnail, rests on the bottom of a glass Petri dish, which the scientists set on the stage of a confocal microscope.
This isn’t just any zebra fish embryo. It has been genetically engineered to carry three mutations: one that makes it immobile and another two that make all its blood vessels fluoresce green and all its red blood cells fluoresce red. Watkins and St Croix are using these fish to screen possible new drugs for treating hypertension, so they want to see how the embryo’s vasculature changes when they bathe it in different compounds. Using an objective lens specially designed to collect large amounts of light at long distances and at high resolution—crucial for imaging a relatively large, living specimen rather than just a plate of cells or tissue—the duo captures 1,000 images per second.
“These physiological responses are happening so quickly, we can’t even see them on [our] computer screen because the screen cannot refresh quickly enough,” Watkins says.
The human visual system can resolve images coming in at about 25 hertz; many computer screens detect flickering at about 100 hertz (though speeds vary quite a bit); but the cameras Watkins works with capture movies at about 1,000-1,600 hertz. “We have to collect blind, and then do the analysis,” he says.
Watkins, the director of the University of Pittsburgh’s Center for Biologic Imaging (CBI), presides over a sprawling, 6,500-squarefoot suite that houses about 30 microscopes. Between them, these devices can perform close to any feat of imaging achievable today: from three-dimensional views of single molecules to the unfolding of physiological events in the cells of living, breathing organisms. Using genetically encoded fluorescent probes, researchers can simultaneously track the activity of five or six different proteins over time.
It’s a striking contrast to the system for detecting fluorescent labels in biological tissue set up by Lans Taylor four decades ago. What was then a state-of-the-art system consisted of a recently declassified night-vision camera hooked up to a Commodore 128-kilobyte computer and a videotape recorder.
Taylor—now on the faculty at Pitt—is the scientist who planted the seed of imaging innovation in Pittsburgh. (He directs Pitt’s Drug Discovery Institute and serves as Allegheny Foundation Professor of Computational and Systems Biology.) Taylor jerry-rigged the Commodore setup when he was a young assistant professor of cell biology at Harvard University. During his PhD work a few years earlier, when he was studying the dynamics of cell motility in amoebas, he realized that fluorescent reagents provided a specific and sensitive way to track cells across space and time. At least, they did sometimes—the fluorescent dyes he used had a lot of problems, like toxicity to cells and chemical instability. Immunologists in the 1940s had learned how to conjugate fluorescent dyes to antibodies, providing a huge boost to the field of immunohistochemistry. But efforts to harness the power of fluorescence for functional studies of the cell were few and far between. By the time Taylor started his own lab in 1974, he was developing his own fluorescent reagents and building imaging systems that could detect the low levels of light emitted. He had an inkling that fluorescence imaging would hit it big as a fundamental technique in the life sciences.
“It became clear when I was still at Harvard in the late 1970s that this was a field that was going to grow,” he says, “and that it would require the integration of biology, chemistry, physics, and computer science.”
Taylor’s hunch about the promise of fluorescence planted a vision in his mind, and he began looking for an institution that was interested in bringing him on to build a center to pull the needed expertise together. The idea struck a chord with Richard Cyert, the late president of Carnegie Mellon University. “President Cyert saw it as a bridge between computer science and engineering—where they had strengths—and biology, where they wanted to grow,” says Taylor.
It’s hard to overstate the prescience of Taylor and a handful of like-minded scientists. Today, fluorescent probes and detection systems comprise a booming, multibillion dollar industry. Open almost any life sciences–related journal to almost any page and you’ll see an array of fluorescence-based colors crisply delineating minute cellular structures or disease markers. Fluorescent probes can be engineered to illuminate specific proteins or cell types, as well as to act as sensors that register changes in physiological phenomena over time. Fluorescence imaging has been used to sequence the human genome, to illuminate cell-signaling pathways, and to guide cancer surgeons. A trio of researchers in 2008 won the Nobel Prize in Chemistry for the discovery and development of the green fluorescent protein, a probe which can be cloned into the genomes of experimental animals and cells. But the sheer number and variety of fluorescent probes and sensors have provided a vast tool kit to biologists seeking to explore almost any question in the life sciences. The imaging center that Taylor launched at CMU in 1982, and its partner imaging center, founded by Watkins at the University of Pittsburgh nine years later, have played a major part in laying the foundation for this methodological revolution. These centers continue to drive innovation in fluorescence imaging technology.
One of Taylor’s first recruits in 1982 to CMU’s Center for Fluorescence Research was Alan Waggoner, a chemist from Amherst College in Massachusetts. Like Taylor, Waggoner had caught the fluorescence bug in the early 1970s. His interest had been piqued by a colleague at Yale University, neurophysiologist Lawrence Cohen, who wanted to find a way to detect electrical signals in neurons, not by poking them with electrodes but just by looking down a microscope. Waggoner synthesized thousands of dyes before he hit on one that fluoresced upon changes in voltage and was relatively stable and nontoxic to cells.
The collaboration was Waggoner’s first experience working with a class of dyes called cyanine dyes. They were the workhorses of the photography industry—developed in the early 1900s and used to increase the range of wavelengths that form an image in photographic film. They were quite bright and photostable. “There was a huge literature on these dyes, but nobody had ever thought much about applying them as labels for biological detection,” he says. “I thought that the cyanine dyes could be modified to make them fluorescent labeling agents.”
When Taylor brought Waggoner aboard at CMU, he asked the chemist to lead the development of multicolored reagents, labels, and probes. Taylor himself would take charge of inventing better fluorescence detection and imaging systems. Then they’d apply these tools to biological problems. That suited Waggoner just fine. He continued his work with cyanine dyes, fiddling with the chemistry to enhance their brightness and make them even more impervious to bleaching under the light of a microscope bulb. The group devised other probes, as well—both fluorescent analogues (fluorescent and trackable versions of molecules of interest) and sensors that could detect cellular processes. On the instrumentation side, Taylor and Frederick Lanni, another early recruit to the center, created the so-called standing wave fluorescence microscope, the forerunner of today’s super-resolution microscope platforms. The group also hatched several imaging techniques, including one called ratiometric imaging, which allowed researchers to quantify intracellular calcium levels, pH, or other biochemical activities by using a probe that fluoresces to different degrees at two different wavelengths.
As CMU’s center continued to grow and innovate, the University of Pittsburgh in 1991 recruited Simon Watkins to start its own imaging center. By then, the field of digital imaging microscopy had progressed to the point where commercial systems using solid state cameras were available, like the Multimode Microscope developed at CMU (see caption on p. 28). However, many biologists continued to use film.
“To take a picture on a fluorescence microscope with film, you’d focus on the image and then press the button and hold it for 15 seconds. And then when you looked at it, the dyes were bleached. And then you’d get the film processed and hope there was an image on it. Isn’t that nuts?” says Watkins.
“Everything was slow, everything was dead, and there was no genetically encoded anything.”
One advance that changed the status quo was confocal microscopy, a technique invented in the 1950s but commercialized just four years before Watkins came to Pitt. Instead of collecting all of the light emitted by a specimen, confocal microscopy uses a pinhole to reject all but the light in focus. Excluding the flare from fluorescence outside of that region dramatically boosted image quality. By the mid-1990s, this technology had flooded the life sciences world, and Pitt was at the front of the wave. Watkins’ facility had acquired four confocal microscopes and was becoming an important resource for scientists at Pitt, as well as at other institutions.
As CMU’s imaging center continued to flourish with the help of a National Science Foundation Science and Technology Center grant, Taylor and Waggoner were beginning to feel the lure of the industry world. In 1991, right after Waggoner patented CyDyes, his line of cyanine dyes, the duo decided to spin off a company, Biological Detection Systems (BDS). That company commercialized the dyes as well as the semi-automated imaging system that Taylor had pioneered; BDS was acquired in 1995 by Amersham (another imaging mecca, now part of GE Healthcare), primarily because the four cyanine dyes could separately detect four nucleotides and thus be used in early DNA sequencers. In addition, the dyes were widely deployed in live-cell studies because they allowed researchers to see chemical and molecular events in the context of time and space within cells.
And: “We could actually image and look, and they wouldn’t bleach,” says Watkins.
With BDS sold, Taylor’s feet got itchier. The way he saw it, fluorescence imaging needed to undergo the same kind of revolution that gene sequencing technology was experiencing. Researchers had been able to manually sequence genes since the 1970s, but in order to undertake an endeavor like the Human Genome Project, which launched in 1990, the process needed to be taken completely out of human hands. Similarly, Taylor says, back then it could take days after loading images to complete an analysis with available software. Yet, to track spatial and temporal dynamics in cells, “we needed to go from the kind of human interactive semi-automated microscopes that we were dealing with in the mid-1990s to fully automated,” he says. “I decided that although technologically we could have done this at Carnegie Mellon, this was really an industrial task.” So while his wife chalked up his departure from academia to a midlife crisis, Taylor set off to launch a second company, Cellomics, in 1996, which created high content screening. This technology automated imaging of cells and small organisms for drug discovery and development. After that, he launched two more companies.
Apart from a brief sabbatical in the UK at Amersham, Waggoner stayed on at CMU; a few years later he began working on a new concept for making modular biosensors that could be designed to follow specific proteins within the cell with unprecedented spatial and temporal resolution. In 2003, the NIH had announced an initiative to fund a network of five multidisciplinary research centers around the country that would develop novel technologies for studying protein function within cellular pathways and networks. Waggoner spoke with Watkins and other investigators at Pitt about applying—an idea that Arthur S. Levine, an MD and Pitt’s senior vice chancellor for the health sciences as well as the John and Gertrude Petersen Dean of the School of Medicine, wholeheartedly encouraged.
“Art just said, ‘Well, Alan, why don’t you go ahead and see if you can put together a proposal and get this thing going—involve some of the University of Pittsburgh people,’” Waggoner recalls. “And that’s what we did.” The relationship between CMU and Pitt was cemented when they received a $13.1 million grant in 2006.
What Waggoner, Watkins, and their colleagues produced within that framework is powerful technology that can detect an enormous variety of cellular processes in real time. These biosensors consist of a dye called a fluorogen, which fluoresces only in the presence of a protein fragment engineered to activate it, called a fluorogen-activating protein, or FAP. When the two bind, the FAP essentially stabilizes the chemical shape of the fluorogen in a way that allows it to fluoresce. “A simple way to think about it is, if you catch a butterfly, its wings can no longer flap,” says Marcel Bruchez, a PhD associate professor of chemistry and biological sciences at CMU who designed the system with Waggoner and Pitt’s St Croix, an assistant professor of environmental and occupational health in the Graduate School of Public Health and associate director of the CBI.
“By catching them in a protein, the previously flopping movement of the electron orbits that make up the dye molecule is suppressed,” Bruchez says. “Held in that rigid protein environment, the dye can emit light when you shine light on it.”
The researchers kept the project moving with weekly informal conversations held at a rotating list of eating and drinking establishments located near the two campuses. The division of labor went like this: CMU researchers would do the chemical fiddling to make novel sensors. Watkins, St Croix, and their crew would test the sensors out in different cell types to show that they worked and then would channel the interest of biologists who might benefit from them. “People walk into Simon’s office and say, ‘I’m trying to figure this out; how can I do it?’” says Bruchez. “He is really good at mastering the biology that’s required to address these problems.”
FAP technology has tremendous specificity. For example, it’s possible to design sensors that don’t pass through cell membranes, and thus specifically detect a protein found only on the outside of a cell; simultaneously, a different color probe can track that same protein within a cell. Along these lines, one application for FAPs is to track the density of proteins called G-protein-coupled receptors (GPCRs) at the cell membrane. GPCRs are a class of molecules mediating many diverse health-related processes. They are targets for about a third of all pharmaceutical drugs on the market, and they accomplish their assigned cellular tasks by communicating with other proteins from a seat in the cell membrane. The FAP assay can quickly screen for GPCR activity by determining how many such proteins have been recruited to the membrane, making it a valuable tool in drug discovery.
Raymond Frizzell, PhD professor of cell biology and director of the cystic fibrosis research center at Pitt, has been using FAPs for cell-surface protein detection as he screens for novel drugs to treat the disease. Cystic fibrosis is caused by mutations in the CFTR protein, one of which blocks the protein’s transport to the cell surface. First, Frizzell’s group used FAPs attached to CFTR to characterize how efficiently correctors, a class of small molecules designed to correct the function of the protein, brought CFTR to the cell surface.
“We could detect very clearly that the protein only got to the cell surface when we used certain correctors or combinations of correctors,” Frizzell says.
Then, they tested those molecules on cells taken from the lungs of cystic fibrosis patients and grown in a culture dish. Those that got to the cell surface membrane in the first assay were the ones that worked best in the cultured cells, too, validating the use of FAPs to find potential drugs. Frizzell’s group is now setting up a high-throughput assay that will use FAPs to screen for more effective corrector compounds.
But FAPs’ real superpowers lie in their ability to detect all sorts of physiological changes, such as membrane potential, calcium concentration, pH, or redox state—not just in cells, but in living animals, like the zebra fish Watkins and St Croix have been screening (which was genetically engineered by Pitt’s Beth Roman, PhD assistant professor of biological sciences).
“I think this [technique] is going to show us a lot of biology that has been hidden in the cell-based experiments that we’ve used for almost all of our basic assays,” says Bruchez. As he, Waggoner, and Watkins continue to refine the physiological sensors, Michael Tsang, a PhD associate professor of developmental biology, is breeding thousands of zebra fish that will express genetically engineered proteins that make FAP dyes light up in response to changes in calcium concentration. If that works, other sensors will follow.
There are already probes that do that, says Waggoner, but they have big limitations. “Those probes diffuse to wherever they want to be in the cell,” he says—which makes it impossible to pin down where the action is taking place.
By targeting proteins specific to the mitochondria, the endoplasmic reticulum, or the cell surface, for example, you can take a reading right there, or follow the signal wherever it goes.
“Then we can ask, What happens in the normal developmental pathway when these dyes come on? And if we manipulate the system, do we see any changes in the morphology of the embryo? ” says Tsang. “With these new tools [you’ll] see these changes happening instantaneously—not just in one cell type or one tissue type, but in the context of a whole organism.”
If the technique works, it could give a breathtakingly intimate look at how the ebbs and flows of one cellular mechanism affect another. Does the way that calcium waves are propagated in the heart affect that organ’s morphology? Does a neuron firing in one part of the brain affect free radical release elsewhere?
In his role as head of drug discovery for Pitt, Taylor now focuses his efforts on new biosensors and ways of studying activities within many cells or organisms instead of just one at a time. The veteran imager predicts a luminous future for fluorescence technology as researchers gradually master approaches to 3-D imaging in humans, tissue-engineered models, and live animal–techniques.
“I would say there won’t be any molecules or biochemical events within cells that we won’t have the ability to make a sensor for,” Taylor says. And just as FAPs and other fluorescent technologies have shown us fundamental biological processes in cells, other innovations in microscopy are also transforming the field. Just five years ago, the resolution microscopes could achieve was stuck between 200 and 500 nanometers; today, sophisticated super-resolution platforms can achieve a resolution of 20-100 nanometers. (Some get as low as 5 nanometers.)
Of course, there’s plenty of work left to do. At present, researchers can only see a few millimeters into living tissue with a microscope.
“It would be great if we could have high-contrast imaging multiple centimeters into animals,” says Waggoner. Two other items on his wish list: dyes with infinite photostability (that never fall apart or create reactive oxygen species which poison cells, no matter how much light they are hit with) and dyes with ever-sharper absorption and emission ranges, so that each one gives a narrow and completely isolatable signal.
Although the NIH grant that formally intertwined the imaging efforts at CMU and Pitt is now in its sunset phase, there is no sign that the two groups plan to wind down their collective activities any time soon. Says Waggoner: “It’s really best when we work together.”
Joe Miksch contributed to this story.
A Murderous Glow
Most fluorescent dyes act as markers or sensors, heralding the presence of a protein or a physiological event. Now there’s a way to bring fluorescence into the action with a dye ominously called KillerRed.
For more than a decade, Pitt’s Li Lan, an assistant professor of microbiology and molecular genetics, has been studying mammalian cell response to DNA damage. Until recently, she used either a blast from an ultraviolet laser or an enzyme called I-SceI to induce such damage. But it’s impossible to pinpoint where exactly on a chromosome a laser hits, and the enzyme, while more precise, creates a cleaner and more artificial break in the DNA than what happens in nature—specifically, in the midst of the production of reactive oxygen species (ROS).
Enter KillerRed, created by Russian scientists, which changes its structure and releases ROS as it absorbs light. It can also be genetically encoded to target a specific location on a chromosome. “KillerRed,” Lan realized, “could combine the benefits of the I-SceI system and the laser system.” The potent dye could help researchers understand a crucial cellular process.
“Every hour, the DNA in each cell of your body undergoes more than 5,000 breaks,” Lan says. Most are quickly repaired, but people in whom that process is impaired accumulate mutations that can lead to cancer and other diseases.
Lan and colleagues figured out a way to fuse KillerRed with a protein that increases gene transcription at a particular location, generating DNA-damaging molecules there. (Her collaborators include CMU’s Marcel Bruchez as well as the School of Medicine’s Robert Sobol, Bennett Van Houten, and Arthur S. Levine, Petersen Dean and senior vice chancellor for the health sciences.)
Last November, Lan published her first paper using the technique, which showed that damaged DNA is repaired differently depending on whether chromosomes are packed tightly, as they normally are, or unwound, as they become when cells divide.
“It’s a big question in the field, but we have not had a good way of analyzing how it happens,” says Lan. Until now. —AK
Images Copyright © Lan et al. 2013. Published by Oxford University Press.