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

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.

[1]  Tobias M. P. Hartwich,et al.  Video-rate nanoscopy using sCMOS camera- specific single-molecule localization algorithms , 2013 .

[2]  Ignacio Izeddin,et al.  PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking. , 2012, Optics express.

[3]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[4]  C. Mackay,et al.  Photon counting strategies with low-light-level CCDs , 2003, astro-ph/0307305.

[5]  Bernd Rieger,et al.  Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs. , 2018, The Journal of chemical physics.

[6]  M. Gustafsson,et al.  Phase‐retrieved pupil functions in wide‐field fluorescence microscopy , 2004, Journal of microscopy.

[7]  Michael W. Davidson,et al.  Video-rate nanoscopy enabled by sCMOS camera-specific single-molecule localization algorithms , 2013, Nature Methods.

[8]  X. Zhuang,et al.  Whole cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution , 2008, Nature Methods.

[9]  Keith A. Lidke,et al.  Simultaneous multiple-emitter fitting for single molecule super-resolution imaging , 2011, Biomedical optics express.

[10]  M. Lakadamyali,et al.  Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate , 2014, Nature Methods.

[11]  P. Annibale,et al.  Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking , 2011, PloS one.

[12]  Thierry Blu,et al.  LEAP: Looking beyond pixels with continuous-space EstimAtion of Point sources , 2017 .

[13]  Xiaowei Zhuang,et al.  Fast compressed sensing analysis for super-resolution imaging using L1-homotopy. , 2013, Optics express.

[14]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[15]  J. Lippincott-Schwartz,et al.  Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure , 2009, Proceedings of the National Academy of Sciences.

[16]  Suliana Manley,et al.  Quantitative evaluation of software packages for single-molecule localization microscopy , 2015, Nature Methods.

[17]  Nicolas Olivier,et al.  FALCON: fast and unbiased reconstruction of high-density super-resolution microscopy data , 2014, Scientific Reports.

[18]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[19]  Y. Okada,et al.  A multi‐emitter fitting algorithm for potential live cell super‐resolution imaging over a wide range of molecular densities , 2018, Journal of microscopy.

[20]  Mike Heilemann,et al.  SuReSim: simulating localization microscopy experiments from ground truth models , 2016, Nature Methods.

[21]  C. Zimmer,et al.  QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ , 2010, Nature Methods.

[22]  Patrick Fox-Roberts,et al.  Local dimensionality determines imaging speed in localization microscopy , 2017, Nature Communications.

[23]  Yaron M Sigal,et al.  A high-density 3D localization algorithm for stochastic optical reconstruction microscopy , 2012, Optical Nanoscopy.

[24]  S. Hess,et al.  Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples , 2008, Nature Methods.

[25]  Hanben Niu,et al.  3D localization of high particle density images using sparse recovery. , 2015, Applied optics.

[26]  Martin Ovesný,et al.  High density 3D localization microscopy using sparse support recovery. , 2014, Optics express.

[27]  Suliana Manley,et al.  Live intracellular super-resolution imaging using site-specific stains. , 2013, ACS chemical biology.

[28]  Jerry Chao,et al.  A Software Framework for the Analysis of Complex Microscopy Image Data , 2010, IEEE Transactions on Information Technology in Biomedicine.

[29]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[30]  J. Bewersdorf,et al.  Biological Insight from Super-Resolution Microscopy: What We Can Learn from Localization-Based Images. , 2018, Annual review of biochemistry.

[31]  Gilles Aubert,et al.  Erratum: A Continuous Exact ℓ0 Penalty (CEL0) for Least Squares Regularized Problem , 2016, SIAM J. Imaging Sci..

[32]  Lucien E. Weiss,et al.  Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions , 2015, Nano letters.

[33]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[34]  Suliana Manley,et al.  3D high-density localization microscopy using hybrid astigmatic/ biplane imaging and sparse image reconstruction. , 2014, Biomedical optics express.

[35]  Michael P. Hobson,et al.  A Stochastic Model for Electron Multiplication Charge-Coupled Devices – From Theory to Practice , 2013, PloS one.

[36]  Hazen P Babcock,et al.  Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines , 2016, Scientific Reports.

[37]  J. Sibarita,et al.  Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy , 2013, PloS one.

[38]  Suliana Manley,et al.  Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging , 2015, PloS one.

[39]  S. Holden,et al.  DAOSTORM: an algorithm for high- density super-resolution microscopy , 2011, Nature Methods.

[40]  Benjamin Recht,et al.  The alternating descent conditional gradient method for sparse inverse problems , 2015, 2015 IEEE 6th International Workshop on Computational Advances in Multi-Sensor Adaptive Processing (CAMSAP).

[41]  Samuel J. Lord,et al.  Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function , 2009, Proceedings of the National Academy of Sciences.

[42]  Guy M. Hagen,et al.  ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging , 2014, Bioinform..

[43]  Ricardo Henriques,et al.  Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations , 2016, Nature Communications.

[44]  Mark Bates,et al.  Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy , 2008, Science.

[45]  Dylan T Burnette,et al.  Bayesian localisation microscopy reveals nanoscale podosome dynamics , 2011, Nature Methods.

[46]  Ulf Matti,et al.  Optimal 3D single-molecule localization in real time using experimental point spread functions , 2018, Nature Methods.

[47]  Sean Quirin,et al.  Super-resolution photon-efficient imaging by nanometric double-helix point spread function localization of emitters (SPINDLE). , 2012, Optics express.

[48]  S. Weiss,et al.  Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI) , 2009, Proceedings of the National Academy of Sciences.

[49]  Chenglong Xia,et al.  Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes , 2012, Proceedings of the National Academy of Sciences.

[50]  Yuejie Chi,et al.  Super-resolution image reconstruction for high-density 3D single-molecule microscopy , 2016, 2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI).