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Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Nature Photonics (2024)Cite this article

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Abstract

In this study, we integrate a single-photon detector array into a confocal laser scanning microscope, enabling the combination of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy. This unique combination delivers a twofold improvement in lateral localization accuracy for single-molecule localization microscopy (SMLM) and maintains its simplicity. Moreover, the addition of lifetime information from our confocal laser scanning microscope eliminates chromatic aberration, particularly crucial for achieving few-nanometre resolution in SMLM. Our approach, named fluorescence-lifetime image scanning microscopy SMLM, is demonstrated through direct stochastic optical reconstruction microscopy and DNA point accumulation for imaging in nanoscale topography experiments on fluorescently labelled cells, showcasing both resolution enhancement and fluorescence-lifetime multiplexing capabilities.

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Fig. 1: iSMLM principle.
Fig. 2: ISM setup and calibration.
Fig. 3: Single-labelling iSMLM dSTORM results.
Fig. 4: dSTORM-based FL-iSMLM multiplexing.
Fig. 5: DNA-PAINT-based FL-iSMLM multiplexing.

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Data availability

All the raw data are available via GitLab at https://gitlab.gwdg.de/igregor/single-molecule-localization-microscopy-with-image-scanning-microscopy40.

Code availability

Every scrip and code to process the raw data is available via GitLab at https://gitlab.gwdg.de/igregor/single-molecule-localization-microscopy-with-image-scanning-microscopy40.

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Acknowledgements

N.R., J.E. and S.O.R. acknowledge financial support from the Bundesministerium für Bildung und Forschung (BMBF), Germany, via project NG-FLIM (project nos. 13N15327 and 13N15328). J.I.G. acknowledges financial support from the European Union’s Horizon 2021 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 101062508 (project name: SOADOPP). J.E. and J.I.G. acknowledge financial support from the DFG through Germany’s Excellence Strategy EXC 2067/1-390729940. J.E. and O.N. thank the European Research Council (ERC) for financial support via project ‘smMIET’ (grant agreement no. 884488) under the European Union’s Horizon 2020 research and innovation programme. We thank A. Chizhik for providing us with the illustration of our ISM microscope setup, as well as for carefully reading the manuscript.

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Author notes

  1. These authors contributed equally: Niels Radmacher, Oleksii Nevskyi, José Ignacio Gallea.

Authors and Affiliations

  1. Third Institute of Physics—Biophysics, Georg August University, Göttingen, Germany

    Niels Radmacher, Oleksii Nevskyi, José Ignacio Gallea, Ingo Gregor & Jörg Enderlein

  2. Department of Chemistry, University of Oxford, Oxford, UK

    Jan Christoph Thiele

  3. Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany

    Silvio O. Rizzoli

  4. Cluster of Excellence ‘Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells’ (MBExC), Universitätsmedizin Göttingen, Göttingen, Germany

    Silvio O. Rizzoli & Jörg Enderlein

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Contributions

N.R. and O.N. built the setup, performed the microscopy imaging and analysed the data. J.I.G. prepared the samples, performed the microscopy imaging and analysed the data. J.C.T. helped with the implementation of the ISM plug-in for the analysis software. I.G. helped with the setup and experiments. J.E. conceived the idea, worked out the theoretical framework and helped with the data analysis. N.R., O.N., J.I.G., S.O.R. and J.E. wrote the manuscript and did the final editing.

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Correspondence to Jörg Enderlein.

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Nature Photonics thanks Liangyi Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Radmacher, N., Nevskyi, O., Gallea, J.I. et al. Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy. Nat. Photon. (2024). https://doi.org/10.1038/s41566-024-01481-4

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