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Super-resolution optical fluctuation imaging

Nature Photonics volume 19, pages 229–237 (2025)Cite this article

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Abstract

We present a comprehensive review of super-resolution optical fluctuation imaging (SOFI), a robust technique that leverages temporal fluctuations in fluorescence intensity to achieve super-resolution imaging without the need for single-molecule localization. The Review starts with a historical overview of super-resolution microscopy techniques, and then focuses on SOFI’s core principle—the analysis of intensity fluctuations using cumulants to improve spatial resolution. The paper discusses technical challenges, such as photobleaching, blinking kinetics and pixel size limitations, as well as proposing solutions like Fourier upsampling and balanced SOFI to mitigate these issues. Additionally, we discuss potential advancements in the field, including the integration of SOFI with other super-resolution modalities like structured illumination microscopy and image scanning microscopy, and the application of SOFI in cryo-fluorescence microscopy and quantum emitter-based imaging. This paper aims to serve as an essential resource for researchers interested in utilizing SOFI for high-resolution imaging in diverse biological applications.

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Fig. 1: Principle of SOFI.
Fig. 2: OTF and PSF of SOFI.
Fig. 3: Pixel size problem.

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Acknowledgements

N.R. and J.E. acknowledge financial support from the Bundesministerium für Bildung und Forschung (BMBF) of Germany via project NG-FLIM (project no. 13N15327). 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). I.Gligonov acknowledges funding from the International Max Planck Research School for Physics of Biological and Complex Systems and by the European Union via the HORIZON–MSCA–2022–DN ‘Improving BiomEdical diagnosis through LIGHT-based technologies and machine learning—BE-LIGHT’ (grant agreement no. 101119924–BE-LIGHT). J.E. and J.I.G. acknowledge financial support from the DFG through Germany’s Excellence Strategy EXC 2067/1390729940. S.B., O.N., N.R. and J.E. 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.

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Authors and Affiliations

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

    Samrat Basak, Alexey Chizhik, José Ignacio Gallea, Ivan Gligonov, Ingo Gregor, Oleksii Nevskyi, Niels Radmacher, Roman Tsukanov & Jörg Enderlein

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

    Jörg Enderlein

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  1. Samrat Basak
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J.E. wrote the manuscript. A.C. and J.E. generated all the figures. S.B., J.I.G., I. Gregor, O.N. and R.T. proofread the final version of the manuscript, and checked the correctness of all citations. N.R. performed the measurements for Fig. 3c. I. Gligonov checked all the theoretical equations in the manuscript.

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Correspondence to Jörg Enderlein.

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Basak, S., Chizhik, A., Gallea, J.I. et al. Super-resolution optical fluctuation imaging. Nat. Photon. 19, 229–237 (2025). https://doi.org/10.1038/s41566-024-01571-3

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