Confocal Fluorescence Imaging of Photosensitized DNA Denaturation in Cell Nuclei

Authors

  • Tytus Bernas,

    1. Laboratory of Confocal Microscopy and Image Analysis, Department of Biophysics, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland
    2. University of Silesia, Department of Plant Anatomy and Cytology, Faculty of Biology and Protection of Environment, Katowice, Poland
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  • Elikplimi K. Asem,

    1. Purdue University, Department of Basic Medical Sciences, West Lafayette, IN
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  • J. Paul Robinson,

    1. Purdue University, Department of Basic Medical Sciences, West Lafayette, IN
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  • Peter R. Cook,

    1. Oxford University, Sir William Dunn School of Pathology, Oxford, UK
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  • Jurek W. Dobrucki

    Corresponding author
    1. Laboratory of Confocal Microscopy and Image Analysis, Department of Biophysics, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland
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  • Posted on the website on 10 May 2005

*To whom correspondence should be addressed: Laboratory of Confocal Microscopy and Image Analysis, Department of Biophysics, Faculty of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. Fax: +48 12/664 6902; e-mail: dobrucki@mol.uj.edu.pl

ABSTRACT

The double-stranded helical structure of DNA is maintained in part by hydrogen bonds between strands and by stacking interactions between adjacent purine and pyrimidine bases in one strand. The transition (denaturation) from a double-stranded (ds) to a single-stranded (ss) form can be induced in isolated DNA or fixed cells by exposure to elevated temperatures, alkali or acids, aprotic or nonpolar solvents or some drugs. We report here that DNA denaturation can occur in situ in cell nuclei as a result of interaction between light and an intercalated dye, acridine orange or ethidium bromide. This DNA photodenaturation was probed using metachromatic properties of acridine orange and imaged by fluorescence confocal microscopy. Furthermore, an empirical kinetic model was developed to separate changes of acridine orange luminescence intensities caused by photobleaching from those that were a result of DNA denaturation. We investigated the influence of oxygen on these phenomena and propose a mechanism by which photodenaturation may occur.

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