Nitric oxide, an antioxidant primarily known for its role as a messenger in cellular signaling events, also is generated by cells to accomplish a variety of other tasks. Macrophages, for example, create it to eliminate bacteria and other foreign particles that they encounter. Biologists who study the effects of nitric oxide on living tissues are interested in clearly identifying the presence of the molecule within cells. However, current imaging methods, including chemiluminescence, electron paramagnetic resonance spectrometry and amperometry, have low spatial resolution and often are costly. Fluorescence microscopy can image nitric oxide in biological contexts with suitable spatiotemporal resolution; however, commonly used fluorescent nitric oxide sensors are unable to monitor the molecule directly. For several years, Stephen J. Lippard and his colleagues at MIT in Cambridge, Mass., have attempted to find a way to trigger fluorescence directly in the presence of nitric oxide. Using a variety of metal ions — such as iron, ruthenium, cobalt and rhodium — in combination with a metal-bound fluorophore, they found that some metal-based compounds could elicit fluorescence in the presence of nitric oxide, but also in the presence of other substances, even water. Now the group has developed a fluorescent probe — made with copper — that directly and rapidly reacts with nitric oxide, enabling imaging of the molecule in living cells. The researchers — Lippard, Mi Hee Lim and Dong Xu — report their findings in the July issue of Nature Chemical Biology. They created the probe by combining a fluorescein-based ligand with CuCl2. In the resulting complex, the fluorescence of the modified fluorescein was quenched. But when nitric oxide was introduced, the compound immediately exhibited an approximately elevenfold increase in fluorescence. The researchers have not measured the precise speed of the reaction yet but, according to Lippard, it is in the range of milliseconds. They recorded the fluorescence emissions with spectrophotometers from Hitachi High Technologies Corp. of Tokyo and from Photon Technology International Inc. of Birmingham, N.J., finding that the maximum emission was ~530 nm. Importantly, introducing nitric oxide to a copper-free version of the fluorescein ligand resulted in only an ~1.5-fold increase in fluorescence, indicating that the copper is indispensable for fluorescence enhancement. The fluorescence occurs because the nitric oxide triggers a reduction of the Cu(II) to Cu(I), which forms NO+. Subsequently, the NO+ reacts with the fluorescein-ligand compound, dissociating the copper from the compound and turning on the fluorescence of the fluorescein. “We were concerned that the resulting nitrosated molecules might be toxic,” Lippard said, “but that turned out not to be the case for the cells we examined, at least under our conditions.” The scientists determined that the technique provides a detection limit of 5 nM for nitric oxide. According to Lippard, the group would like to improve upon that, although no one knows exactly what the ultimate limit should be. To test the viability of copper-fluorescein complexes for imaging nitric oxide in living cells, the investigators imaged human neuroblastoma cells, using both the copper-based construct and commercially available o-diaminofluorescein diacetate (DAF-2 DA), which typically reacts solely with the oxidation products of nitric oxide. In neuroblastoma cells, nitric oxide is produced by constitutive nitric oxide synthase (cNOS) that is activated by a common calcium-based cell-signaling sequence that begins with the introduction of estrogen. Imaging the cells with inverted microscopes from Nikon and from Zeiss, they found that the copper-fluorescein compound induced a nitric-oxide-dependent fluorescence enhancement of about fourfold within the five minutes required to bring the cells from the tissue culture room to the microscope (Figure 1). Conversely, when DAF-2 DA was used in the same estrogen-treated cells, there was an ~1.8-fold increase in fluorescence after 30 minutes. Figure 1. Using a novel complex of copper and a metal-bound fluorescein, researchers have imaged nitric oxide produced in neuroblastoma cells via the introduction of estrogen. At t = 0 (top row), the copper-fluorescein particles are present, but not estrogen. Five minutes after introduction of estrogen, fluorescence increased fourfold (second row). The third and fourth rows represent t = 10 min and t = 15 min, respectively. Images courtesy Stephen J. Lippard and Mi Hee Lim. Just as cNOS originates nitric oxide in neuroblastoma, inducible nitric oxide synthase (iNOS) produces nitric oxide inside macrophages. And, as with neuroblastoma cells, the researchers found that the copper-fluorescein compound could monitor the expected slow increase in fluorescence over a span of 12 hours in almost every region of the macrophages following treatment with lipopolysaccharide and interferonγ. They also mixed neuroblastoma and macrophage cells in the same culture dish, finding that, by adding estrogen, they could use the sensor to preferentially detect fluorescence in the nitric oxide produced by the neuroblastoma’s cNOS (Figure 2). The scientists believe that this technique could be useful for providing information about which cells produce nitric oxide within heterogeneous tissue samples as well as for determining the timing and placement of intracellular events involving nitric oxide. Further tests indicated that the copper-fluorescein compound is not toxic to either the neuroblastoma or the macrophage cells, and Lippard’s group has arranged with a chemical company to produce an assay kit based on the technique. Figure 2. Mixing neuroblastoma and macrophage cells in the same culture dish, the investigators found that they could use the copper-based sensor to preferentially detect fluorescence in the nitric oxide produced by the neuroblastoma. The researchers are already planning improvements on the compound. “It is not reversible,” Lippard said, “and it is not trappable.” Trappability — the ability to keep the copper-fluorescein particles inside the cells — would be useful for live-cell studies of nitric oxide and cell signaling processes in which the samples must be consistently presented with nutrients that could wash the fluorescent compound away. They also are working toward a version of the compound that can be activated by wavelengths in the near-infrared range, which can penetrate skin tissue better than the 530-nm wavelength used experimentally. “Wavelengths greater than 700 nm might allow us to image what nitric oxide is doing in vivo,” Lippard said.