As the technology and capabilities of microscopy continue to advance, an area within the medical school’s Center for Cellular and Molecular Imaging (CCMI) is incorporating the latest types of equipment. Known for nearly two decades as the “confocal” facility, it houses microscopes that enable the visualization and imaging of fixed and living tissues that contain fluorescent probes. “More than 1,000 publications have emerged from the facility,” says Michael H. Nathanson, MD, PhD, Gladys Phillips Crofoot Professor of Medicine (Digestive Diseases) and professor of cell biology, director of the Yale Liver Center, and director of CCMI. “Seventy to 80 grants cite us as an available resource at any given time.” Nathanson anticipates getting even busier, as new devices that incorporate such technologies as swept field, light sheet, Airyscan, PALM/STORM, and STED 3X super resolution take their place at CCMI alongside the original confocal and multi-photon microscopes.

When confocal microscopes became widely available in the second half of the 20th century, Nathanson says, they were “fantastic,” far surpassing the resolution that wide field microscopy delivered. “I’d say now we’re in the middle of another revolution pushing the limits beyond what you can get just from confocal or even multi-photon microscopy.”

The facility’s gated (time-interval isolating) STED super-resolution instrument “allows us to see things about five times better than you theoretically can see them based on the diffraction limit,” Nathanson says. A swept field device that arrived last fall incorporates confocal technology, but also “can collect images up to 500 or maybe even 1,000 frames per second with good resolution,” a revelation when investigators want to follow very rapid processes involving live specimens. “We can see with high spatial resolution very transient events, even in subcellular regions.”

An Airyscan device that CCMI acquired early this year offers higher resolutions than standard confocal microscopy with great ease of use. It already is popular among investigators, according to Nathanson, who anticipates that yet another 2018 acquisition—the PALM/STORM super-resolution system—will produce levels of resolution that eclipse those of the now two-year-old gated STED. That is how quickly the field is moving.

Mustafa K. Khokha, MD, associate professor of pediatrics (critical care) and of genetics, studies fundamental questions about what happens in the womb to cause heart malformations that later afflict the babies he treats in his clinical practice. His investigations in frogs focus on processes that include the function of cilia, small projections from the surfaces of certain cells whose rhythmic beating can have an important impact on fluid flow as the fetal heart takes shape. The high-resolution images of live specimens by the Bruker swept field instrument, captured at hundreds of frames per second, contain detail that was unattainable in the past.

Khokha points to a monitor on his desk. “These are cilia in the frog embryo. We can watch them beating in two colors so we can see the tips. That is nothing we could do before.” [See the moving image here] Cilia can form and vanish within a day, and a single formation of cilia during gestation may be the difference between a healthy heart and one that is malformed for life. “We are pushing the biology to a point that those kinds of details make a big difference,” Khokha says.

Various details call for various microscopes, and Khokha uses the full array of instruments in the CCMI core. At times, he needs speed and sensitivity more than maximum resolution. Or, he may need an instrument that scans rapidly but is not designed for live imaging. Sometimes he waits to figure out the best method. “By having them [all] in a core, my lab can go down there and say, ‘Okay, for this experiment, this might be the best scope,’ and I’ll take an image there,” Khokha says.

Elizabeth A. Jonas, MD, professor of internal medicine (endocrinology) and of neuroscience, is preparing for a series of brain investigations at the CCMI core. They involve explaining mechanisms that govern hearing and identifying the sources of sounds, part of a larger project where, she says, “we are asking questions about how the synapse [the interface where signals are passed between neurons] is plastic. In other words, how does it change when you have to learn something.”

Up until now, her lab has worked in cell culture, and already the Airyscan 880 has revealed structural detail that was unknown before. “Now, we can see how actin and synaptic vesicles are really positioned in the synapse,” Jonas says. “Before, it was just a bunch of fuzz.” Jonas anticipates being able to do high-resolution imaging deep within brain slices, and in structures like the basal ganglia that sit in the center of the brain and are not on the top layer or the cortical tissue. “We want to be able to image those deep layers in live animals with dyes or fluorescent markers,” Jonas says.

The vivid images that Yale investigators are starting to see are the products of advanced software, laser technology, high-speed cameras, and massive amounts of data that these microscopes generate and process, according to Joerg Bewersdorf, PhD, professor of cell biology and of biomedical engineering, and one of the world’s leading experts in microscope development. Seeing exquisite images, so long the Holy Grail of investigators, is now “just the first step,” he says. The data have value far beyond creating the images themselves, essential as the images are, he explains. “Modern biological research has shifted away from pure description or showing an example image. People expect statistics now—quantification.” Each, he says, is also a check on the other. A high-resolution image might contradict poorly analyzed or incomplete data, and solid data might show that an image is aberrant.

At the same time, Yale is moving into an emerging field called correlative microscopy, which combines big datasets derived from microscopes that visualize specimens in vastly different ways, in order to bridge inherent knowledge gaps among the varied methods. The timing for that move is exquisite, says Bewersdorf, with Yale’s acquisition last year of the Titan Krios cryo-electron microscope representing one set of techniques, and now the CCMI core’s advances in the other. “We are in an excellent position now,” says Bewersdorf, “because we have instruments from both sides, the latest and greatest to bring these together. You will not find a lot of facilities with this number of high-end instruments at one location.”