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Journal of Histochemistry and Cytochemistry, Vol. 47, 183-196, February 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Phosphorescent Platinum/Palladium Coproporphyrins for Time-resolved Luminescence Microscopy

Richard R. de Haasa, Rob P.M. van Gijlswijka, Erik B. van der Tolb, Jacky Veuskensc, Hilde E. van Gijsseld, Roeline B. Tijdensd, Jan Bonneta, Nico P. Verwoerda, and Hans J. Tankea
a Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Leiden
b Department of Organic Chemistry, University of Amsterdam, Amsterdam
c Institute for Molecular Cell Biology, University of Amsterdam
d Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, University of Leiden, Leiden, The Netherlands

Correspondence to: Richard R. de Haas, Lab. for Cytochemistry and Cytometry, Dept. of Molecular Cell Biology, Leiden Univ. Medical Center; Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase). (J Histochem Cytochem 47:183–196, 1999)

Key Words: metalloporphyrins, time-resolved luminescence microscopy, phosphorescence, autofluorescence, FISH, bleaching, tyramide signal amplification


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