OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 19, Iss. 8 — Apr. 11, 2011
  • pp: 7020–7033

Measuring localization performance of super-resolution algorithms on very active samples

Steve Wolter, Ulrike Endesfelder, Sebastian van de Linde, Mike Heilemann, and Markus Sauer  »View Author Affiliations

Optics Express, Vol. 19, Issue 8, pp. 7020-7033 (2011)

View Full Text Article

Enhanced HTML    Acrobat PDF (996 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.

© 2011 OSA

OCIS Codes
(100.6640) Image processing : Superresolution
(180.2520) Microscopy : Fluorescence microscopy
(350.4800) Other areas of optics : Optical standards and testing

ToC Category:
Image Processing

Original Manuscript: December 21, 2010
Revised Manuscript: February 23, 2011
Manuscript Accepted: February 27, 2011
Published: March 29, 2011

Virtual Issues
Vol. 6, Iss. 5 Virtual Journal for Biomedical Optics

Steve Wolter, Ulrike Endesfelder, Sebastian van de Linde, Mike Heilemann, and Markus Sauer, "Measuring localization performance of super-resolution algorithms on very active samples," Opt. Express 19, 7020-7033 (2011)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316, 1153–1158 (2007). [CrossRef] [PubMed]
  2. P. Kner, B. B. Chhun, E. R. Griffis, L. Winoto, and M. G. L. Gustafsson, “Super-resolution video microscopy of live cells by structured illumination,” Nat. Methods 6, 339–342 (2009). [CrossRef] [PubMed]
  3. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging Intracellular Fluorescent Proteins at Nanometer Resolution,” Science 313, 1642–1645 (2006). [CrossRef] [PubMed]
  4. S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91, 4258–4272 (2006). [CrossRef] [PubMed]
  5. B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319, 810–813 (2008). [CrossRef] [PubMed]
  6. M. Heilemann, S. van de Linde, M. Sch¨ttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. 47, 6172–6176 (2008). [CrossRef]
  7. J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Nat. Acad. Sci. U.S.A. 106, 8107–8112 (2009). [CrossRef]
  8. R. Wombacher, M. Heidbreder, S. van de Linde, M. P. Sheetz, M. Heilemann, V. W. Cornish, and M. Sauer, “Live-cell super-resolution imaging with trimethoprim conjugates,” Nat. Methods 7, 717–719 (2010). [CrossRef] [PubMed]
  9. H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5, 417–423 (2008). [CrossRef] [PubMed]
  10. T. Klein, A. Löschberger, S. Proppert, S. Wolter, S. van de Linde, and M. Sauer, “Live-cell dstorm with snap-tag fusion proteins,” Nat. Methods 8, 7–9 (2011). [CrossRef]
  11. N. Bobroff, “Position measurement with a resolution and noise-limited instrument,” Rev. Sci. Instrum. 57, 1152–1157 (1986). [CrossRef]
  12. M. K. Cheezum, W. F. Walker, and W. H. Guilford, “Quantitative comparison of algorithms for tracking single fluorescent particles,” Biophys. J. 81, 2378–2388 (2001). [CrossRef] [PubMed]
  13. R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise Nanometer Localization Analysis for Individual Fluorescent Probes,” Biophys. J. 82, 2775–2783 (2002). [CrossRef] [PubMed]
  14. K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods 7, 377–381 (2010). [CrossRef] [PubMed]
  15. C. Shannon, “Communication in the Presence of Noise (reprinted),” Proc. IEEE 72, 1192–1201 (1984). [CrossRef]
  16. S. van de Linde, S. Wolter, M. Heilemann, and M. Sauer, “The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging,” J. Biotechnol. 149, 260–266 (2010). [CrossRef] [PubMed]
  17. T. Cordes, M. Strackharn, S. W. Stahl, W. Summerer, C. Steinhauer, C. Forthmann, E. M. Puchner, J. Vogel-sang, H. E. Gaub, and P. Tinnefeld, “Resolving single-molecule assembled patterns with superresolution blink-microscopy,” Nano Lett. 10, 645–651 (2010). [CrossRef]
  18. M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5, 527–529 (2008). [CrossRef] [PubMed]
  19. S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5, 155–157 (2008). [CrossRef] [PubMed]
  20. M. B. J. Roeffaers, B. F. Sels, H. Uji-i, F. C. De Schryver, P. A. Jacobs, D. E. De Vos, and J. Hofkens, “Spatially resolved observation of crystal-face-dependent catalysis by single turnover counting,” Nature 439, 572–575 (2006). [CrossRef] [PubMed]
  21. M. B. J. Roeffaers, G. De Cremer, J. Libeert, R. Ameloot, P. Dedecker, A.-J. Bons, M. Bückins, J. A. Martens, B. F. Sels, D. E. De Vos, and J. Hofkens, “Super-resolution reactivity mapping of nanostructured catalyst particles,” Angew. Chem. Int. Ed. 48, 9285–9289 (2009). [CrossRef]
  22. H. Bornfleth, K. Sätzler, R. Eils, and C. Cremer, “High-precision distance measurements and volume-conserving segmentation of objects near and below the resolution limit in three-dimensional confocal fluorescence microscopy,” Journal of Microscopy 189, 118–136 (1998). [CrossRef]
  23. S. Holden, S. Uphoff, and A. Kapanidis, “Crowded-field photometry increases maximum super-resolution imaging density by an order of magnitude,” Nat. Methods (2010). Manuscript submitted. [PubMed]
  24. S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann, and M. Sauer, “Real-time computation of subdiffraction-resolution fluorescence images,” J. Microsc. 237, 12–22 (2010). [CrossRef] [PubMed]
  25. J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Nat. Acad. Sci. U.S.A. 107, 10068–10073 (2010). [CrossRef]
  26. T. Quan, P. Li, F. Long, S. Zeng, Q. Luo, P. N. Hedde, G. U. Nienhaus, and Z.-L. Huang, “Ultra-fast, high-precision image analysis for localization-based super resolution microscopy,” Opt. Express 18, 11867–11876 (2010). [CrossRef] [PubMed]
  27. D. A. Grossman and O. Frieder, Information Retrieval: Algorithms and Heuristics , The Kluwer International Series of Information Retrieval (Springer, Box P.O. 17, 3300 AA Dordrecht, The Netherlands, 2004), 2nd ed.
  28. A. TechnologyiXon camera manual (Andor Technology, 7 Millennium Way, Springvale Business Park, Belfast, BT12 7AL, NORTHERN IRELAND, 2008).
  29. M. Galassi, J. Davies, J. Theiler, B. Gough, G. Jungman, M. Booth, and F. Rossi, Gnu Scientific Library: Reference Manual (Network Theory Ltd., 2003).
  30. M. Matsumoto and T. Nishimura, “Mersenne twister: a 623-dimensionally equidistributed uniform pseudo-random number generator,” ACM Trans. Model. Comput. Simul. 8, 3–30 (1998). [CrossRef]
  31. S. Ehrich, “Error bounds for Gauss–Kronrod quadrature formulae,” Math. Comp. 62, 295–304 (1994). [CrossRef]
  32. D. M. Thomann, “Algorithms for detection and tracking of objects with super-resolution in 3d fluorescence microscopy,” Ph.D. thesis, ETH Zürich (2003).
  33. R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7, 339–340 (2010). [CrossRef] [PubMed]
  34. T. A. Laurence and B. A. Chromy, “Efficient maximum likelihood estimator fitting of histograms,” Nat Meth 7, 338–339 (2010). [CrossRef]
  35. N. A. Frost, H. Shroff, H. Kong, E. Betzig, and T. A. Blanpied, “Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines,” Neuron 67, 86 – 99 (2010). [CrossRef] [PubMed]
  36. U. Endesfelder, S. van de Linde, S. Wolter, M. Sauer, and M. Heilemann, “Subdiffraction-resolution fluorescence microscopy of myosin-actin motility,” Phys. Chem. Chem. Phys. 11, 836–840 (2010).

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited