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The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

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Abstract

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.

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Acknowledgments

This research was financially supported by the Slovenian Research Agency: research grant P-0371 and in part by the united States Army Medical Research Materiel Command (USAMRMC) Grant10782106, and Worcester Polytechnic Institute. We thank Klemen Ziberna for all the help with the manuscript preparation.

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  1. Matija Veber

    Present address: Educell Ltd., Letališka cesta 33, 1000, Ljubljana, Slovenia

Authors and Affiliations

  1. Blood Transfusion Centre of Slovenia, Šlajmerjeva 6, 1000, Ljubljana, Slovenia

    Mojca Jež, Matija Veber & Primož Rožman

  2. Department of Biomedical Engineering, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA, 01609, USA

    Tuba Bas & Raymond Page

  3. Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, 1000, Ljubljana, Slovenia

    Andrej Košir

  4. Department of Biology and Biotechnology, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA, 01609, USA

    Tanja Dominko

  5. Bioengineering Institute, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA, 01609, USA

    Tanja Dominko & Raymond Page

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  1. Mojca Jež
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Correspondence to Primož Rožman.

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M. Jež and T. Bas contributed equally to this work.

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Jež, M., Bas, T., Veber, M. et al. The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem. Histochem Cell Biol 139, 195–204 (2013). https://doi.org/10.1007/s00418-012-1039-8

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  • DOI: https://doi.org/10.1007/s00418-012-1039-8

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