Increasing the luminescence of lanthanide complexes

Abstract

This review compares the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species that can be used as labels for cytometry. A series of luminescent lanthanide ion macrocyclic complexes, Quantum Dyes Ò , which do not release or exchange their central lanthanide ion, do accept energy transfer from ligands, and are capable of covalent binding to macromolecules, including proteins and nucleic acids, is described and their properties are discussed.Two methods are described for increasing the luminescence intensity of lanthanide ion complexes, which intrinsically is not as high as that of standard fluorophores or quantum dots. One method consists of adding a complex of a second lanthanide ion in a micellar solution (columinescence); the other method produces dry preparations by evaporation of a homogeneous solution containing an added complex of a second lanthanide ion or an excess of an unbound antenna ligand. Both methods involve the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect as the mechanism for the luminescence enhancement. q 2006 International Society for Analytical Cytology Key terms: Quantum Dye; RETEL; lanthanide; macrocycle; luminescence; columinescence; time-delayed; europium; terbium Only one article published in Cytometry, by Seveus et al.(1), has described in detail the use of a lanthanide ion complex as a luminescent label for an antibody, and this article, published in 1992, dealt primarily with the instrumentation for time-gated microscopy. Thus, it is appropriate for a special issue of Cytometry to include a focused review of this class of luminescent labels. Extensive reviews of the clinical and other uses of lanthanide complexes have previously been published by Hemmil€ a and coworkers (2-4).This review presents a comparison of the spectral properties, relative sizes, and essential chemical features of lanthanide complexes and other narrow emitting labels. It also provides a critical description of two related approaches that can be used to overcome the comparatively low molar extinction coefficients (molar absorptivities) of lanthanide ion complexes: 1) the columinescence effect, where the luminescence of a lanthanide complex is increased in a micellar solution by energy transfer from a complex of a non-emitting lanthanide to a complex of an emitting lanthanide, and 2) the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect (5) where the energy transfer occurs in the solid state.A companion Technical Note (6) describes the experimental aspects of the RETEL effect (5), which resulted in a major increase of the luminescence intensity of a specific type of lanthanide macrocycles, the Quantum Dyes Ò . This increase in luminescence facilitates the use of the Quantum Dyes as labels, either with fluorescent microscopes conventionally illuminated by a Mercury-Xenon (Hg-Xe) arc or-when it is helpful to eliminate contamination from the emissions of conventional fluorophoreswith new cost-effective time-gating instrumentation.The emissions...

Increasing lanthanide luminescence by use of the RETEL effect

“…(5) show that the RETEL effect requires either a solid state or an aqueous emulsion (columinescence) (17)(18)(19). The increase in luminescence mediated by the Gd 31 ion (or by La 31 and Y 31 ) does not occur in a true (clear) solution (6).…”

“…Since the usable excitation period for time-gated imaging is proportional to the half-life of the emitting species, increasing the excitation period from the 2 ls provided by a flash-lamp, such as a Hamamatsu L4634, to the 1-2 ms provided by the LED is sufficient to overcome the advantage of the much greater molar extinction coefficient of quantum dots and conventional fluorochromes relative to LnMacs (6). In this context, it should be noted that the half-life of quantum dots has been FIG.…”

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

“…This equipment has the capacity to obtain unique fluorescence spectra of specific dye molecules that will increase our understanding of biological fluorescence emission process (9)(10)(11)(12)(13)(14)(15)(16). The popularity of this new field in biology has been described in a special issue of Cytometry that includes both review articles and specific application articles (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). To insure that this equipment is functioning properly, new and improved tests have been developed to evaluate spectral performance on these instruments (1,2,5,6).…”

“…http://probes.invitrogen. com/media/pis/mp36909.pdf) with peak emission spectra vary between 500 and 700 nm, and the Vallarino-Leif Europium macrocycle Quantum Dye bead with defined peaks at 595, 619, and 685 nm, and a FWHM below 10 nm (21,29). These beads can be used with all confocal and non-confocal spectral systems having the correct excitation light source.…”

“…It is beneficial to have a bead that has the characteristics of the Europium Quantum Dye with its chemically defined emission peak and very small FWHM (4,21,34). The combination of the position of the peaks and the width of the histograms are parameters that can be used to determine if the spectroscopic system is working correctly.…”