Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software


With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities—2D, astigmatic 3D, biplane 3D and double-helix 3D—and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.

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Fig. 1: Summary of SMLM challenge simulations.
Fig. 2: Leaderboards for each competition modality, at low and high spot density.
Fig. 3: Comparison of 3D software performance.
Fig. 4: Super-resolved images of software results for simulated and real competition datasets.

Data availability

Simulated competition datasets are available at, together with the parameters used to generate the data. The ground-truth list of simulated molecule positions for each competition dataset remains secret to allow the software challenge to remain continuously open to new submissions. However, ground-truth data are available for the simulated training datasets. Source data for Figs. 14 and for Supplementary Figs. 47, 19, 20 and 22 are available online.

Code availability

All software is available at

Change history

  • 16 May 2019

    In the version of this paper originally published, Figure 4a contained errors that were introduced during typesetting. The bottom 11° ThunderSTORM image is an xz view but was incorrectly labeled as xy, and the low x-axis value in the four line profiles was incorrectly set as –60 instead of –50. These errors have been corrected in the PDF and HTML versions of the paper.


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The authors acknowledge the following funding sources: a Newcastle University Research Fellowship and a Wellcome Trust and Royal Society Sir Henry Dale Fellowship grant number 206670/Z/17/Z to S.H.; a European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program, Grant Agreement number 692726 to D.S., T.A.P., and M.U.; UK BBSRC grants BB/M022374/1, BB/P027431/1, BB/R000697/1 grant and MRC grants MC-UU-12018/2, MR/K015826/1 to R.H.; ERC grant CoG-724489, CellStructure to J.R.; FranceBioImaging infrastructure ANR-10-INBS-04 to J.-B.S.; National Institutes of Health grant 1R15GM128166-01 to G.M.H.; and NSF SBIR grants 1353638, 1534745 to Double Helix LLC. We thank R. Piestun at University of Colorado for providing double-helix PSF phase mask designs to Double Helix LLC. We thank all the localization microscopy challenge participants for their contribution: H. Babcock (3D-DAOSTORM, Cspline, L1H); F. Hauser (3D STORM Tools); S. Watanabe (3D-WTM,WTM); N. Boyd (ADCG); J. Min, K. Jin and J.C. Ye (ALOHA, FALCON); H. Rouault (B-recs); E. Soubies (CEL0-STORM); A. Speiser, S. Turagas and J. Macke (DECODE); A. von Diezmann, C. Bayas and W.E. Moerner (Easy-DHPSF); T. Vomhof and J. Reichel (FIRESTORM); H. Pan (LEAP); A. Wheeler (Localizer); Z.-l. Huang and Y. Wang (MaLiang); J. Chao, R. Velmurugan, A.V. Abraham and R.J. Ober (MIATool); H. Deschout (mlePALM); T. Pengo (Octane, PeakSelector); Y.-n. Wang (PALMER); A. Herbert (PeakFit); K. Martens and J. Hohlbein (pSMLM-3D); L. Li (QC-STORM); R. Henriques (QuickPALM); G. Tamas and J. Sinko (RainSTORM); S. Wolter and M. Sauer (RapidSTORM); M. Kirchgessner and F. Gruell (SFP Estimator); Y. Li and J. Ries (SMAP); H. Ikoma (SMfit); A. Loot, A. Valdmann, M. Eltermann, M. Kree and M. Pärs (SMolPhot); Y.J. Jung, A. Barsic, R. Piestun and N. Fakhri (SOLAR_STORM); A. Archetti (STORMChaser); M. Ovesny, G. Hagen and P. Krizek (ThunderSTORM); J. Huang (TVSTORM); A. Kechkar, C. Butler and J.-B. Sibarita (WaveTracer) and B. Lelandais (ZOLA-3D). We thank the SMLMS 2016 organizers (S. Manley and A. Radenovic, EPFL) for hosting a localization microscopy challenge special session. We also thank Double Helix and Molecular Devices for sponsoring the SMLMS 2016 special session. The sponsors had no input or influence on the research.

Author information

D.S. and S.H. conceived and coordinated the study. D.S., S.H., T.-A.P., A. Archetti, H.B., S.C., A.W., G.M.H., R.H., T.L., T.P., and J.-B.S. designed the study. S.H., A. Agrawal, R.H., and J.-B.S. collected experimental PSFs. D.S., T.-A.P., S.H., and T.L. wrote simulation code. B.R. shared unpublished software. D.S. generated simulated datasets. J.R. shared experimental STORM data. A.H., J.R., J.C., and R.V. provided feedback and quality control on simulations and analysis methods. T.-A.P. carried out the assessment of software performance. T.-A.P., D.S., and S.H. analyzed and interpreted the results. D.S., H.B., R.O., B.R., G.M.H., J.-B.S., J.R., R.H., M.U., and S.H. directed research. S.H., D.S., and T.-A.P. wrote the manuscript with feedback from all other authors.

Correspondence to Daniel Sage or Seamus Holden.

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A. Agrawal is an employee of Double Helix LLC, USA.

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Sage, D., Pham, T., Babcock, H. et al. Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software. Nat Methods 16, 387–395 (2019).

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