With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. 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 single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.@en

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.@en