Increasing lanthanide luminescence by use of the RETEL effect

Background: Luminescent lanthanide complexes produce emissions with the narrowest‐known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion review, Leif et al., Cytometry 2006;69A:767–778, described a new technique for the enhancement of lanthanide luminescence, the Resonance Energy Transfer Enhanced Luminescence (RETEL) effect, which increases luminescence and is compatible with standard slide microscopy. Methods: The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the nonluminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand or the sodium salt of TTFA in ethanol solution, the EuMac‐labeled sample was allowed to dry. Both a conventional arc lamp and a time‐gated UV LED served as light sources for microscopic imaging. The emission intensity was measured with a CCD camera. Multiple time‐gated images were summed with special software to permit analysis and effective presentation of the final image. Results: With the RETEL effect, the luminescence of the EuMac‐streptavidin conjugate increased at least six‐fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac‐anti‐5BrdU conjugate was subsequently attached. Time‐gated images showed the long‐lived EuMac luminescence but did not show the short‐lived DAPI fluorescence. Imaging of DNA‐synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes®, as molecular diagnostics for cytological and histopathological microscopic imaging. © 2006 International Society for Analytical Cytology