Although lanthanides have been used for biological imaging, they are somewhat limited for use in living tissue because they must be excited with ultraviolet light. Now researchers at the University of Lyon, at the Institut Albert Bonniot in Grenoble and at the University of Joseph Fourier in St. Martin d’Hères, all in France, have developed a europium complex that can be excited using the two-photon antenna effect in the near-infrared spectral range, which may help overcome these limitations.Elements in the lanthanide series, which include neodymium, europium, ytterbium and terbium, have several qualities that make them highly desirable for fluorophores. They have sharp transitions with large Stokes shifts located in the visible or the near-infrared ranges, and they are insensitive to oxygen. Lanthanides also can be caged and have long excited-state lifetimes, ranging from micro- to milliseconds. The researchers, led by Olivier Maury, developed a functionalized tris-dipicolinate EuIII complex that has many of the features desirable for a biological fluorophore: It is soluble and stable in aqueous media, is luminescent, has a long fluorescence lifetime, and it has a large two-photon absorption cross section. Maury said that the most challenging aspect of developing the fluorophore was designing to so many different requirements. The complex needed a high quantum yield, which required an optimum energy transfer between the antenna ligand and the metal. It needed high two-photon absorption but also good stability and solubility as well as conservation of the photophysical properties in water. “In the literature, is it possible to find some examples of europium complexes that fulfill one or the other requirement but, to the best of my knowledge, never all requirements,” he said. In particular, he added, it is difficult to build a complex that presents good linear and nonlinear optical properties in water.The researchers recorded the two-photon-excitation spectrum in the 700- to 900-nm range using a femtosecond Ti:sapphire laser to confirm that the two-photon-excitation wavelength almost perfectly doubles the UV excitation wavelength centered at 318 nm. The two-photon cross section measured 92 GM at 700 nm. Tests of the new fluorophore on T24 cancer cells fixed with ethanol clearly showed the fluorophore, which localized in the perinuclear region. Researchers have developed a europium-based complex that is soluble and stable in water, has a long fluorescence lifetime and has a large two-photon-absorption cross section. Courtesy of Olivier Maury, University of Lyon.“This compound is proof of concept that it is possible to carry out two-photon imaging microscopy with lanthanide complexes,” Maury said. He added that the sharp transition of the lanthanide complex improves the signal-to-noise ratio and that the excitation at 760 nm and emission at 616 nm both lie within the biological window. He also was careful to point out that the researchers’ experiments were conducted with commercially available equipment. “Furthermore, the very long luminescence lifetime of such a complex opens the way for the development of time-gated two-photon microscopy that should further improve the signal-to-noise ratio.” The researchers plan to optimize the complex’s photophysical properties in water, to increase the compatibility of the existing complexes with the biological media and to develop which part of the cell the complex will target. In the longer term, he said that the group is looking at designing a lanthanide-based fluorescent immunoassay for practical applications in biology.Journal of the American Chemical Society, Feb. 6, 2008, pp. 1532-1533.