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J Fluoresc. 2014 Nov;24(6):1791-801. doi: 10.1007/s10895-014-1468-y. Epub 2014 Oct 14.

UV-induced spectral shift and protonation of DNA fluorescent dye Hoechst 33258.

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Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387, Kraków, Poland.


DNA-bound Hoechst 33258 is readily excited with UV light and emits blue fluorescence, however, upon exposure to UV, the dye undergoes photobleaching as well as photoconversion to a blue-excited green-emitting form. We demonstrate that the UV-generated green-emitting form of Hoechst 33258 exhibits spectral properties very similar to the form of the dye that can be obtained by subjecting it to an acidic environment (pH 0.5-3.0). We also demonstrate that exposure of Hoechst 33258 to UV light (or hydrogen peroxide) leads to generation of the protonated (1+, 2+, 3+ and possibly the 4+) forms of the dye. Photoconversion of Hoechst 33258 has recently been exploited in single molecule localisation microscopy, thus understanding photophysics of this process can facilitate further development of high resolution optical imaging.

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