<title>Methods to increase the luminescence of lanthanide (III) macrocyclic complexes</title>

Simultaneous detection of both a Eu(III) and a Sm(III) Quantum DyeTM is now possible because the enhanced luminescence (cofluorescence) of the Eu(III) and Sm(III) macrocycles occurs in the same solution and with excitation at the same wave- lengths between 350 to 370 nm. Since DAPI is also excited between 350 to 370 nm, it is possible to use common excitation optics and a single dichroic mirror for measuring two molecular species and DNA. The narrow emissions of these macrocycles can be detected with negligible overlap between themselves or with DAPI-stained DNA. This will permit precise pixel by pixel ratio measurements of the Eu(III) macrocycle to Sm(III) macrocycle, and of each macrocycle to DNA. This technology should be applicable to antibodies, FISH, comparative genomic hybridization, and chromosome painting. Cofluorescence of the Tb(III)-macrocycle has also been obtained under different conditions. The luminescence of these lanthanide macrocycles can be observed with conventional fluorescence instrumentation at previously unattainable low levels. Thus, it will be possible to employ narrow bandwidth lanthanide luminescent tags to identify three molecular species with a conventional microscope.