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Multi-target spectrally resolved fluorescence lifetime imaging microscopy

Nature Methods volume 13, pages 257262 (2016) | Download Citation

Abstract

We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

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Acknowledgements

We thank T. Krüger and M. Alsheimer (Department of Cell and Developmental Biology, Biozentrum Universität Würzburg, Würzburg, Germany) for providing plasmids and antibodies. This work was supported by the Biophotonics Initiative of the German Bundesministerium für Bildung und Forschung (BMBF grants 13N10432 and 13N12781 to T.N., A.L. and M.S.).

Author information

Affiliations

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

    • Thomas Niehörster
    • , Anna Löschberger
    •  & Markus Sauer
  2. Drittes Physikalisches Institut, Georg-August-Universität, Göttingen, Germany.

    • Ingo Gregor
    •  & Jörg Enderlein
  3. PicoQuant GmbH, Berlin, Germany.

    • Benedikt Krämer
    • , Hans-Jürgen Rahn
    • , Matthias Patting
    •  & Felix Koberling

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Contributions

T.N., A.L., I.G., M.S. and B.K. designed experiments. T.N., A.L., I.G., B.K., H.-J.R., M.P. and F.K. generated and processed data. T.N., A.L., J.E., I.G., B.K. and M.S. wrote, edited and approved the final draft of manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Markus Sauer.

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    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Tables 1 and 2, and Supplementary Notes 1–3

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    Supplementary Software

    sFLIM software

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DOI

https://doi.org/10.1038/nmeth.3740

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