Data Analysis for Single-Molecule Localization Microscopy

  • Steve Wolter
  • Thorge Holm
  • Sebastian van de Linde
  • Markus Sauer
Part of the Neuromethods book series (NM, volume 86)


We review single-molecule localization microscopy techniques with a focus on computational techniques and algorithms necessary for their use. The most common approach to single-molecule localization, Gaussian fitting at positions pre-estimated from local maxima, is illustrated in depth and techniques for two- and three-dimensional data analysis are highlighted. After an introduction explaining the principle requirements of single-molecule localization microscopy, we discuss and contrast novel approaches such as maximum likelihood estimation and model-less fitting. Finally, we give an overview over the existing, scientifically available software and show how these techniques can be combined to quickly and easily obtain super-resolution images.

Key words

Super-resolution imaging Localization microscopy dSTORM PALM Data analysis rapidSTORM 



We would like to thank the Biophotonics Initiative of the BMBF for financial support (Grants #13N11019 and #13N12507).


  1. 1.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645PubMedCrossRefGoogle Scholar
  2. 2.
    Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91(11):4258–4272PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (storm). Nat Methods 3(10):793–795PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47(33):6172–6176CrossRefGoogle Scholar
  5. 5.
    van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6(7):991–1009, ISSN1754-2189PubMedCrossRefGoogle Scholar
  6. 6.
    Fölling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S, Eggeling C, Hell SW (2008) Fluorescence nanoscopy by ground-state depletion and single molecule return. Nat Methods 5(11):943–945, ISSN 1548-7091PubMedCrossRefGoogle Scholar
  7. 7.
    Vogelsang J, Cordes T, Forthmann C, Steinhauer C, Tinnefeld P (2009) Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy. Proc Natl Acad Sci U S A 106(20):8107–8112PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Lemmer P, Gunkel M, Weiland Y, Müller P, Baddeley D, Kaufmann R, Urich A, Eipel H, Amberger R, Hausmann M, Cremer C (2009) Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range. J Microsc 235(2):163–171PubMedCrossRefGoogle Scholar
  9. 9.
    Dedecker P, Hotta J-I, Flors C, Sliwa M, Uji-i H, Roeffaers MBJ, Ando R, Mizuno H, Miyawaki A, Hofkens J (2007) Subdiffraction imaging through the selective donut mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa. J Am Chem Soc 129(51):16132–16141PubMedCrossRefGoogle Scholar
  10. 10.
    Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11):780PubMedCrossRefGoogle Scholar
  11. 11.
    Gustafsson MGL (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A 102(37):13081–13086PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Heilemann M, Margeat E, Kasper R, Sauer M, Tinnefeld P (2005) Carbocyanine dyes as efficient reversible single-molecule optical switch. J Am Chem Soc 127(11):3801–3806PubMedCrossRefGoogle Scholar
  13. 13.
    Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94(10):108101PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW, Tsien RY (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5(6):545–551, ISSN 1548-7091PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Cheezum MK, Walker WF, Guilford WH (2001) Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys J 81(4):2378–2388PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin v walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065PubMedCrossRefGoogle Scholar
  18. 18.
    Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution imaging with small organic fluorophores. Angew Chem Int Ed 48(37):6903–6908CrossRefGoogle Scholar
  19. 19.
    van de Linde S, Wolter S, Heilemann M, Sauer M (2010) The effect of photoswitching kinetics and labeling densities on superresolution fluorescence imaging. J Biotechnol 149(4):260–266, ISSN 0168-1656PubMedCrossRefGoogle Scholar
  20. 20.
    van de Linde S, Krstić I, Prisner T, Doose S, Heilemann M, Sauer M (2011) Photoinduced formation of reversible dye radicals and their impact on superresolution imaging. Photochem Photobiol Sci 10:499–506PubMedCrossRefGoogle Scholar
  21. 21.
    Endesfelder U, van de Linde S, Wolter S, Sauer M, Heilemann M (2010) Subdiffraction resolution fluorescence microscopy of myosin-actin motility. Chemphyschem 11(4):836–840PubMedCrossRefGoogle Scholar
  22. 22.
    Wombacher R, Heidbreder M, van de Linde S, Sheetz MP, Heilemann M, Cornish VW, Sauer M (2010) Livecell super-resolution imaging with trimethoprim conjugates. Nat Methods 7(9):717–719, ISSN 1548-7091PubMedCrossRefGoogle Scholar
  23. 23.
    Owen DM, Rentero C, Rossy J, Magenau A, Williamson D, Rodriguez M, Gaus K (2010) Palm imaging and cluster analysis of protein heterogeneity at the cell surface. J Biophotonics 3(7):446–454, ISSN 1864-0648PubMedCrossRefGoogle Scholar
  24. 24.
    Henriques R, Lelek M, Fornasiero EF, Valtorta F, Zimmer C, Mhlanga MM (2010) QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat Methods 7(5):339–340, ISSN 1548-7091PubMedCrossRefGoogle Scholar
  25. 25.
    Williamson DJ, Owen DM, Rossy J, Magenau A, Wehrmann M, Gooding JJ, Gaus K (2011) Pre-existing clusters of the adaptor lat do not participate in early T cell signaling events. Nat Immunol 12(7):655–662, ISSN 1529-2908PubMedCrossRefGoogle Scholar
  26. 26.
    Klein T, Löschberger A, Proppert S, Wolter S, van de Linde S, Sauer M (2011) Live-cell dSTORM with SNAP-tag fusion proteins. Nat Methods 8(1):7–9, ISSN 1548-7091Google Scholar
  27. 27.
    Izeddin I, Specht CG, Lelek M, Darzacq X, Triller A, Zimmer C, Dahan M (2011) Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe. PLoS One 6(1):e15611PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Holden SJ, Uphoff S, Kapanidis AN (2011) Daostorm: an algorithm for high-density superresolution microscopy. Nat Methods 8(4):279–280, ISSN 1548-7091PubMedCrossRefGoogle Scholar
  29. 29.
    Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C, Fölling J, Jakobs S, Schönle A, Hell SW, Eggeling C (2010) Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys J 99(8):2686–2694, ISSN0006-3495PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Jones SA, Shim S-H, He J, Zhuang X (2011) Fast, three-dimensional super-resolution imaging of live cells. Nat Methods 8(6):499–505PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Shannon CE (1984) Communication in the presence of noise (reprinted). Proc IEEE 72(9):1192–1201, ISSN 0018-9219CrossRefGoogle Scholar
  32. 32.
    Dertinger T, Colyer R, Iyer G, Weiss S, Enderlein J (2009) Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc Natl Acad Sci U S A 106(52):22287–22292PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Huang F, Schwartz SL, Byars JM, Lidke KA (2011) Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed Opt Express 2(5):1377–1393PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Wolter S, Schüttpelz M, Tscherepanow M, van de Linde S, Heilemann M, Sauer M (2010) Real-time computation of subdiffraction-resolution fluorescence images. J Microsc 237(1):12–22PubMedCrossRefGoogle Scholar
  35. 35.
    Neubeck A, Van Gool L (2006) Efficient non-maximum suppression. In: ICPR’06: proceedings of the 18th international conference on pattern recognition. IEEE Computer Society, Washington, DC, pp 850–855. ISBN 0-7695-2521-0Google Scholar
  36. 36.
    Thomann DM (2003) Algorithms for detection and tracking of objects with super-resolution in 3D fluorescence microscopy. PhD thesis, ETH Zürich.Google Scholar
  37. 37.
    Křížek P, Raška I, Hagen GM (2011) Minimizing detection errors in single molecule localization microscopy. Opt Express 19(4):3226–3235PubMedCrossRefGoogle Scholar
  38. 38.
    Thomann D, Dorn J, Sorger PK, Danuser G (2003) Automatic fluorescent tag localization II: improvement in superresolution by relative tracking. J Microsc 211(Pt 3):230–248, ISSN 0022-2720PubMedCrossRefGoogle Scholar
  39. 39.
    Bobroff N (1986) Position measurement with a resolution and noise-limited instrument. Rev Sci Instrum 57(6):1152–1157CrossRefGoogle Scholar
  40. 40.
    Mlodzianoski MJ, Bewersdorf J (2009) 3D-resolution in FPALM/PALM/STORM. Biophys J 96(3 suppl 1):636–637, ISSN 0006-3495CrossRefGoogle Scholar
  41. 41.
    Aguet F, van de Ville D, Unser M (2005) A maximum-likelihood formalism for sub-resolution axial localization of fluorescent nanoparticles. Opt Express 13:10503–10522PubMedCrossRefGoogle Scholar
  42. 42.
    Stallinga S, Rieger B (2010) Accuracy of the Gaussian point-spread-function model in 2D localization microscopy. Opt Express 18(24):24461–24476PubMedCrossRefGoogle Scholar
  43. 43.
    Baddeley D, Cannell MB, Soeller C (2010) Visualization of localization microscopy data. Microsc Microanal 16(1):64–72PubMedCrossRefGoogle Scholar
  44. 44.
    Zhang B, Zerubia J, Olivo-Marin JC (2007) Gaussian approximations of fluorescence microscope point-spread function models. Appl Optics 46(10):1819–1829CrossRefGoogle Scholar
  45. 45.
    Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810–813, ISSN 1095-9203PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Holtzer L, Meckel T, Schmidt T (2007) Nanometric three-dimensional tracking of individual quantum dots in cells. Appl Phys Lett 90(5):053902CrossRefGoogle Scholar
  47. 47.
    Juette MF, Gould TJ, Lessard MD, Mlodzianoski MJ, Nagpure BS, Bennett BT, Hess ST, Bewersdorf J (2008) Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5(6):527–529, ISSN 1548-7091PubMedCrossRefGoogle Scholar
  48. 48.
    Pavani SRP, Thompson MA, Biteen JS, Lord SJ, Liu N, Twieg RJ, Piestun R, Moerner WE (2009) Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc Natl Acad Sci U S A 106(9):2995–2999PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW, Fetter RD, Hess HF (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci U S A 106(9):3125–3130PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Wolter S, Endesfelder U, van de Linde S, Heilemann M, Sauer M (2011) Measuring localization performance of superresolution algorithms on very active samples. Opt Express 19(8):7020–7033PubMedCrossRefGoogle Scholar
  51. 51.
    Steinhauer C, Forthmann C, Vogelsang J, Tinnefeld P (2008) Superresolution microscopy on the basis of engineered dark states. J Am Chem Soc 130(50):16840–16841PubMedCrossRefGoogle Scholar
  52. 52.
    Cordes T, Strackharn M, Stahl SW, Summerer W, Steinhauer C, Forthmann C, Puchner EM, Vogelsang J, Gaub HE, Tinnefeld P (2010) Resolving single-molecule assembled patterns with superresolution blink-microscopy. Nano Lett 10(2):645–651, PMID:20017533PubMedCrossRefGoogle Scholar
  53. 53.
    van de Linde S, Kasper R, Heilemann M, Sauer M (2008) Photoswitching microscopy with standard fluorophores. Appl Phys B 93(4):725–731CrossRefGoogle Scholar
  54. 54.
    Bates M, Huang B, Dempsey GT, Zhuang X (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317(5845):1749–1753PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    Flors C, Ravarani CNJ, Dryden DTF (2009) Super-resolution imaging of DNA labelled with intercalating dyes. Chemphyschem 10(13):2201–2204PubMedCrossRefGoogle Scholar
  56. 56.
    Weston KD, Carson PJ, DeAro JA, Buratto SK (1999) Single-molecule detection fluorescence of surface-bound species in vacuum. Chem Phys Lett 308:58–64CrossRefGoogle Scholar
  57. 57.
    Vogelsang J, Kasper R, Steinhauer C, Person B, Heilemann M, Sauer M, Tinnefeld P (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed 47(29):5465–5469CrossRefGoogle Scholar
  58. 58.
    Kottke T, van de Linde S, Sauer M, Kakorin S, Heilemann M (2010) Identification of the product of photoswitching of an oxazine fluorophore using Fourier transform infrared difference spectroscopy. J Phys Chem Lett 1(21):3156–3159CrossRefGoogle Scholar
  59. 59.
    Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30(11):1191–1212, ISSN 0891-5849PubMedCrossRefGoogle Scholar
  60. 60.
    Sies H (1999) Glutathione and its role in cellular functions. Free Radic Biol Med 27(9–10):916–921PubMedCrossRefGoogle Scholar
  61. 61.
    York AG, Ghitani A, Vaziri A, Davidson MW, Shroff H (2011) Confined activation and subdiffractive localization enables whole-cell palm with genetically expressed probes. Nat Methods 8(4):327–333, ISSN 1548-7091PubMedCentralPubMedCrossRefGoogle Scholar
  62. 62.
    Hedde PN, Fuchs J, Oswald F, Wiedenmann J, Nienhaus GU (2009) Online image analysis software for photoactivation localization microscopy. Nat Methods 6(10):689–690, ISSN 1548-7091PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Steve Wolter
    • 1
  • Thorge Holm
    • 1
  • Sebastian van de Linde
    • 1
  • Markus Sauer
    • 1
  1. 1.Lehrstuhl für Biotechnologie und BiophysikUniversity of WürzburgWürzburgGermany

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