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.

Advanced Imaging Resource Institute of Genetics and Molecular Medicine University of Edinburgh Edinburgh UK

Biomedical Imaging Group School of Engineering Ecole Polytechnique Fédérale de Lausanne Lausanne Switzerland

Centre for Bacterial Cell Biology Institute for Cell and Molecular Biosciences Newcastle University Newcastle UK

Centre for Cancer Immunology University of Southampton Southampton UK

Department of Biomedical Engineering Texas A and M University College Station TX USA

Department of Imaging Physics Delft University of Technology Delft the Netherlands

Department of Microbial Pathogenesis and Immunology Texas A and M University Health Science Center Bryan TX USA

Department of Molecular and Cellular Medicine Texas A and M University Health Science Center College Station TX USA

Department of Radioelectronics FEE Czech Technical University Prague Czech Republic

Double Helix LLC Boulder CO USA

European Molecular Biology Laboratory Cell Biology and Biophysics Unit Heidelberg Germany

Harvard Center for Advanced Imaging Harvard University Cambridge MA USA

Interdisciplinary Institute for Neuroscience Centre National de la Recherche Scientifique UMR 5297 Bordeaux France

Interdisciplinary Institute for Neuroscience University of Bordeaux Bordeaux France

Istituto Italiano di Tecnologia Genova Italy

Laboratory of Experimental Biophysics École Polytechnique Fédérale de Lausanne Lausanne Switzerland

Laboratory of Nanoscale Biology and Laboratoire d'Optique Biomédicale STI IBI Ecole Polytechnique Fédérale de Lausanne Lausanne Switzerland

MRC Genome Damage and Stability Centre School of Life Sciences University of Sussex Brighton UK

Quantitative Imaging and Nanobiophysics Group MRC Laboratory for Molecular Cell Biology University College London London UK

UCCS Center for the Biofrontiers Institute University of Colorado Colorado Springs CO USA

University of Minnesota Informatics Institute University of Minnesota Twin Cities Minneapolis MN USA

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Nat Methods. 2019 Jun;16(6):561. doi: 10.1038/s41592-019-0453-4. PubMed

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Optimized molecule detection in localization microscopy with selected false positive probability

Imaging Viral Infection by Fluorescence Microscopy: Focus on HIV-1 Early Stage