BioRxiv preprint: Super-resolution microscopy fight club!

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

Daniel SageThanh-An PhamHazen BabcockTomas LukesThomas PengoRamraj VelmuruganAlex HerbertAnurag AgarwalSilvia ColabreseAnn WheelerAnna ArchettiBernd RiegerRaimund OberGuy M. HagenJean-Baptiste SibaritaJonas RiesRicardo HenriquesMichael UnserSeamus Holden


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.