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.

The activation energy of nature-inspired hackmanites was tuned, adapting this material for UV detection.

In this report, we describe luminescence imaging microscopy using five different photoluminescent dyes in a single specimen. We combined the long decay time luminophores, europium(III) chelate, terbium(III) chelate, palladium(II) coproporphyrin, and platinum(II) coproporphyrin, with a green nuclear stain, Syto 25 trade mark, that emits conventional fast decaying fluorescence. The luminescence emissions from the five different luminophores were separated from each other by the differences in spectra and decay times using time-resolved detection. Applicability of this dye-combination for multiparameter analysis of a biological object was verified in a mixed population of peripheral blood leukocytes. Leukocyte cytocentrifugates were incubated in one step with a cocktail of luminophore-conjugated antibodies recognizing neutrophil- and lymphocyte-specific markers, followed by rapid staining with a mixture of nuclear stain and Pt-porphyrin as an eosinophil stain. The results show that multiple luminescent dyes with long decay time can be used together, and in combination with a conventional fluorophore. The separation of the signals of the long decay time labels was distinctive and enabled reliable identification of different leukocyte types, as well as an automated cell count. The long decay time luminophores together with time-resolved luminescence imaging microscopy (TR-LIM) provide a unique tool for studies of simultaneous expression of multiple antigens at the level of a single cell. In comparison with other multiparameter imaging techniques, the described technique offers increased accuracy of results, simplification of preparation procedure, and dramatic shortening of the total processing time. To our knowledge, this is the first time that simultaneous fivefold labeling/staining and analysis in a single specimen has been performed in the field of immunocytochemistry.

No abstract