Studying arterial diseases and therapies just got easier, thanks to a new fluorescence imaging technique that allows researchers to visualize the blood flow of living animals with unprecedented clarity. The Stanford University technique, called near-infrared-II imaging, or NIR-II, involves shining a laser over a living subject — in this case, a mouse — after water-soluble carbon nanotubes have been injected into its bloodstream. The near-infrared wavelength light (about 0.8 µm) causes the specially designed nanotubes to fluoresce at a longer wavelength of 1 to 1.14 µm, which is then detected to determine the blood vessels’ structure. These images of a mouse’s blood vessels show the difference in resolution between traditional near-infrared fluorescence imaging (top) and Stanford University’s new NIR-II technique, which involves shining a laser over a living subject after water-soluble carbon nanotubes have been injected into its bloodstream. Courtesy of Stanford University. The substantially longer wavelengths are the key to the sharp, fine-detailed images: Longer wavelength light scatters less, and thus creates sharper images of the vessels. Additionally, the detector registers less background noise since the body does not produce autofluorescence in this wavelength range. The increase in sharpness compared with that of conventional imaging techniques is akin to wiping fog off your glasses. In addition to providing fine details, the method yields a fast image acquisition rate, allowing researchers to measure blood flow in near real time — a feat previously not possible. “For medical research, it’s a very nice tool for looking at features in small animals,” said chemistry professor Hongjie Dai, who developed the technique with professor of cardiovascular medicine John Cooke and acting assistant professor of cardiothoracic surgery Ngan Huang. “It will help us better understand some vasculature diseases and how they respond to therapy, and how we might devise better treatments.” The technique will be particularly useful in studying arterial disease, such as how blood flow is affected by the arterial blockages and constrictions that cause, among other things, strokes and heart attacks. Because NIR-II can only penetrate, at most, a centimeter into the body, it will not replace other human imaging methods, but it will be powerful enough to study animal models by replacing or complementing x-ray, CT, MRI and laser Doppler techniques. Graduate student Guosong Hong, left, and chemistry professor Hongjie Dai look at the vascular structures in a mouse model of peripheral arterial disease; blood vessels are shown in great detail using their new imaging technique called near-infrared II fluorescence imaging. The technique, developed by the Dai research group in collaboration with the John Cooke group from the Stanford School of Medicine, allows them to see the small biological structures deep inside a body with much greater clarity and higher spatial resolution than current fluorescence-based imaging techniques. Courtesy of L.A. Cicero. The team now plans to explore alternative fluorescent molecules, which will make the technology more easily accepted for use in humans, Dai said. “We’d like to find something smaller than the carbon nanotubes but that emit light at the same long wavelength, so that they can be easily excreted from the body and we can eliminate any toxicity concerns,” he said. The study appeared in Nature Medicine (doi: 10.1038/nm.2995). For more information, visit: www.stanford.edu