PLATINUM PORPHYRINS AS PHOSPHORESCENT LABEL FOR TIME-RESOLVED MICROSCOPY

We investigated phosphorescent metalloporphyrins as potential labels f or time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coe fficient, decay times) the best candidates were selected. Next, we syn thesized antibody and avidin metalloporphyrin conjugates. The optimal F/P ratio with respect to quantum yield, decay time, and retention of biological activity of these immunoreagents was determined. The reagen ts were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of hapten-labeled DNA probes, membrane a ntigens (CD type), and 28S rRNA. All stained samples exhibited bright phosphorescence that could be selectively detected using time-resolved microscopy, especially when glucose/glucose oxidase was added to the embedding medium to deplete oxygen. Applications of time-resolved dete ction of phosphorescent porphyrins in strongly autofluorescent materia l (histological sections) are discussed.