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Photoblueing of organic dyes can cause artifacts in super-resolution microscopy

Nature Methods volume 18, pages 253–257 (2021)Cite this article

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Abstract

Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.

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Fig. 1: Ensemble photoconversion experiments.
Fig. 2: Photoblueing and photowriting with red-absorbing dyes.

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Data availability

All data that support the findings described in this study are available in the manuscript and the related supplementary information, and from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

S.S.M. and M.J.S. were supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (NCI), Center for Cancer Research. We thank the Biophysics Resource in the Structural Biophysics Laboratory, Center for Cancer Research, NCI at Frederick for assistance with liquid chromatography–mass spectrometry analysis. D.A.H., G.B. and M.S. acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG SA829/19-1 and TRR 166 ReceptorLight, project A04) and the European Regional Development Fund (EFRE project, Center for Personalized Molecular Immunotherapy).

Author information

Author notes

  1. These authors contributed equally: Dominic A. Helmerich, Gerti Beliu.

Authors and Affiliations

  1. Department of Biotechnology and Biophysics, Biocenter, Julius Maximilian University of Würzburg, Würzburg, Germany

    Dominic A. Helmerich, Gerti Beliu & Markus Sauer

  2. Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Siddharth S. Matikonda & Martin J. Schnermann

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  1. Dominic A. Helmerich
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  3. Siddharth S. Matikonda
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  4. Martin J. Schnermann
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  5. Markus Sauer
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Contributions

M.S., G.B. and M.J.S. conceived the experiments. D.A.H., G.B. and S.S.M. prepared samples, performed measurements and analyzed data. M.S. wrote the manuscript with input from all authors.

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Correspondence to Markus Sauer.

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The authors declare no competing interests.

Additional information

Peer review information Nature Methods thanks Susana Rocha and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Fluorescence decays and exponential fits of Cy5 before and after irradiation.

The photoconverted (photobleached) product was purified by HPLC. The fluorescence decays were recorded at the emission maxima of 670 and 565 nm, respectively, in PBS, pH 7.4. The lower panels show residual differences between experimental and fitted values. We measured main lifetime components of 0.93 ns with an amplitude > 98% before irradiation and 0.32 ns with an amplitude > 98% for the photoconverted product.

Source data

Supplementary information

Source data

Source Data Fig. 1

Spectroscopic raw data of all figures

Source Data Extended Data Fig. 1

Source data for Extended Data Fig. 1

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Helmerich, D.A., Beliu, G., Matikonda, S.S. et al. Photoblueing of organic dyes can cause artifacts in super-resolution microscopy. Nat Methods 18, 253–257 (2021). https://doi.org/10.1038/s41592-021-01061-2

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