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Journal of Histochemistry and Cytochemistry, Vol. 45, 1279-1292, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

Richard R. de Haasa, Rob P.M. van Gijlswijka, Erik B. van der Tolb, Henry J.M.A.A. Zijlmansa, Tom Bakker-Schuta, Jan Bonneta, Nico P. Verwoerda, and Hans J. Tankea
a Department of Cytochemistry and Cytometry, Leiden University, Leiden, The Netherlands
b Department of Organic Chemistry, University of Amsterdam, Amsterdam, The Netherlands

Correspondence to: Richard R. de Haas, Dept. of Cytochemistry and Cytometry, Faculty of Medicine, Leiden University, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.

We investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized 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 reagents were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of hapten-labeled DNA probes, membrane antigens (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 detection of phosphorescent porphyrins in strongly autofluorescent material (histological sections) are discussed. (J Histochem Cytochem 45:1279-1292, 1997)

Key Words: metalloporphyrin conjugates, time-resolved luminescence microscopy, FISH, autofluorescence, bleaching, tyramide amplification system, phosphorescence


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