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Lanthanides have recently found applications in different fields of biomolecular and medical research. Luminescent lanthanide chelates have created interest mainly due to their unique luminescent properties, such as their long Stokes' shift and exceptional decay times allowing efficient temporal discrimination of background interferences in the assays, such as immunoassays. Recently, new organometallic complexes have been developed giving opportunities to novel applications, in heterogeneous and homogeneous immunoassays, DNA hybridization assays, high-throughput screening as well as in imaging. In addition, encapsulating the chelates into suitable matrix in beads enables the use of new members of lanthanides extending the emission wavelength to micrometer range and decays from a few microseconds to milliseconds. As the luminescence is derived from complicated intra-chelate energy transfer, it also gives novel opportunities to exploit these levels in different types of energy transfer based applications. This review gives a short overview of recent development of lanthanide chelate-labels and discusses in more details of energy levels and their exploitation in new assay formats.

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The synthesis and luminescence properties are reported for 20 different chelates composed of 2,2':6',2"-terpyridine as the energy-absorbing and donating group, Eu"' and Tb"' as the emitting ions, methylenenitrilo(acetic acids) as the stable chelate-forming moieties, and isothiocyanato or (4,6-dichloro-1,3,5-triazin-2-yl)amino groups as the activated moieties for coupling to biomolecules.Introduction. -Time-resolved fluorometry combined with long-lifetime emitting lanthanide chelate labels provides an excellent way of creating highly sensitive label technologies for bioaffinity assays [ 11. A technology based on dissociative fluorescence enhancement [2], Devia@, has gained wide applications in the field of clinical diagnostics in immunoassays [3] and recently also in DNA hybridization assays [4]. In spite of the high sensitivity obtained, the Devia-type of technology is not suited for all applications, such as fluorescence imaging, immunohistochemistry, or in situ hybridization, because after ion dissociation it does not produce spatial information. To use luminescent lanthanide chelates also in in situ assays, new chelate labels need to be developed combining

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased.We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed.A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type I1 collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cer- vix.The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehydefixed, wax-embedded specimens. 0 1992 Wiley-Liss, Inc.