Because intermolecular distances are small, Förster resonance energy transfer (FRET) measurements must be precise to determine whether molecules interact. Using a fluorophore that simultaneously emits two colors and averaging those signals can reduce the error of the measurements. In living cells, new seminaphthofluorone (SNAFR) fluorophores emit white light composed of red, green and violet-blue bands that may enable FRET to be performed with three colors. Robert M. Strongin and colleagues at Louisiana State University in Baton Rouge created the fluorophores and evaluated their spectroscopic properties. They started their synthesis with benzoxanthenes because they exhibit two simultaneous emission bands, enabling two-color FRET. And because near-IR excitation minimizes cellular autofluorescence, they followed another research group’s prediction that variations of existing benzo-xanthenes would absorb near-IR radiation, and the SNAFRs were produced. Researchers created seminaphthofluorones, fluorophores that emit white light and can be excited with various laser lines and viewed with common red, green or blue filter sets. Reprinted with permission of the American Chemical Society. The scientists confirmed that they had created several fluorophores with naphthofluorone frameworks by x-ray crystallography with Mo Kα radiation on a Bruker AXS diffractometer equipped with an Oxford Cryosystems Ltd. cooling system. To study their properties, they collected UV-VIS spectra with a Varian Inc. spectrometer and fluorescence spectra with one from Horiba Jobin Yvon of Edison, N.J. Upon excitation with a 450-W xenon arc lamp, emission passed through a dual monochromator and a Hamamatsu photomultiplier tube upon which 950 V had been applied. When excited at varying pH, overlapping points at which a prototypical SNAFR absorbs light occur at 484, 394, 327 and 304 nm, suggesting that it is suitable for FRET with common filter sets. It can be excited from 260 up to 600 nm in buffer, a range of wavelengths that includes helium-neon, argon-ion and helium-cadmium lasers. It exhibited two pH-sensitive, dual-emission bands, as is the case with other benzoxanthenes. In methanol, it showed no noticeable decrease in photostability for at least 3600 s. These living cells were labeled with a seminaphthofluorone and viewed through a Texas Red filter set. The investigators examined the fluorophores in live cells with an inverted microscope and digital camera, both from Carl Zeiss of Hawthorne, N.Y., as well as with Chroma Technology Corp. filter sets. They demonstrated that they could visualize the fluorophore with ordinary red, blue and green filter sets in living cells. SNAFR readily enters cells and labels the nucleus, possibly because it binds to DNA. It also labels their lipophilic portions, including the endoplasmic reticulum and mitochondria, because it is nonpolar at neutral pH. Strongin hopes that the lipophilic properties of naphthofluorones will enable the molecules to cross the blood-brain barrier. He pointed out that the fluorophores will be readily conjugatable to amino acids after appropriate analogs are synthesized, and that their ability to be used with various filter sets and laser lines may make them useful for confocal microscopy or flow cytometry. He envisions a future for multiplexing applications because they are pH-sensitive and excitable over a broad range of wavelengths. Journal of the American Chemical Society, Nov. 1, 2006, pp. 14081-14092.