Chinese Science Bulletin

, Volume 58, Issue 36, pp 4519–4527

Super-resolution microscopy of live cells using single molecule localization

Invited Review Biophysics


The resolution of conventional light microscopy is insufficient for subcelluar studies. The invention of various super-resolution imaging techniques breaks the diffraction barrier and pushes the resolution limit towards the nanometer scale. Here, we focus on a category of super-resolution microscopy that relies on the stochastic activation and precise localization of single molecules. A diversity of fluorescent probes with different characteristics has been developed to achieve super-resolution imaging. In addition, with the implementation of robust localization algorithms, this family of approaches has been expanded to multi-color, three-dimensional and live cell imaging, which provides a promising prospect in biological research.


resolution super-resolution microscopy single molecules fluorescent probes localization algorithms 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

11434_2013_6088_MOESM1_ESM.pdf (272 kb)
Supplementary material, approximately 271 KB.


  1. 1.
    Hell S W, Wichmann J. Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy. Opt Lett, 1994, 19: 780–782ADSCrossRefPubMedGoogle Scholar
  2. 2.
    Hofmann M, Eggeling C, Jakobs S, et al. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci USA, 2005, 102: 17565–17569ADSCrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Gustafsson M G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc, 2000, 198: 82–87CrossRefPubMedGoogle Scholar
  4. 4.
    Gustafsson M G. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA, 2005, 102: 13081–13086ADSCrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Betzig E, Patterson G H, Sougrat R, et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science, 2006, 313: 1642–1645ADSCrossRefPubMedGoogle Scholar
  6. 6.
    Hess S T, Girirajan T P, Mason M D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J, 2006, 91: 4258–4272CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Rust M J, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (storm). Nat Methods, 2006, 3: 793–795CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Heilemann M, van de Linde S, Schuttpelz M, et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed, 2008, 47: 6172–6176CrossRefGoogle Scholar
  9. 9.
    Thompson R E, Larson D R, Webb W W. Precise nanometer localization analysis for individual fluorescent probes. Biophys J, 2002, 82: 2775–2783CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ober R J, Ram S, Ward E S. Localization accuracy in single-molecule microscopy. Biophys J, 2004, 86: 1185–1200CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Schoen I, Ries J, Klotzsch E, et al. Binding-activated localization microscopy of DNA structures. Nano Lett, 2011, 11: 4008–4011ADSCrossRefPubMedGoogle Scholar
  12. 12.
    Kiel A, Kovacs J, Mokhir A, et al. Direct monitoring of formation and dissociation of individual metal complexes by single-molecule fluorescence spectroscopy. Angew Chem Int Ed, 2007, 46: 3363–3366CrossRefGoogle Scholar
  13. 13.
    Manley S, Gillette J M, Patterson G H, et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods, 2008, 5: 155–157CrossRefPubMedGoogle Scholar
  14. 14.
    Lee S H, Shin J Y, Lee A, et al. Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (palm). Proc Natl Acad Sci USA, 2012, 109: 17436–17441ADSCrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Dempsey G T, Vaughan J C, Chen K H, et al. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods, 2011, 8: 1027–1036CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zhu Z J, Yeh Y C, Tang R, et al. Stability of quantum dots in live cells. Nat Chem, 2011, 3: 963–968CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zhang M, Chang H, Zhang Y, et al. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods, 2012, 9: 727–729CrossRefPubMedGoogle Scholar
  18. 18.
    Shroff H, Galbraith C G, Galbraith J A, et al. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods, 2008, 5: 417–423CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lakadamyali M, Babcock H, Bates M, et al. 3D multicolor super-resolution imaging offers improved accuracy in neuron tracing. PLoS One, 2012, 7: e30826ADSCrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Patterson G, Davidson M, Manley S, et al. Superresolution imaging using single-molecule localization. Annu Rev Phys Chem, 2010, 61: 345–367CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lippincott-Schwartz J, Patterson G H. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol, 2009, 19: 555–565CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Patterson G H, Lippincott-Schwartz J. A photoactivatable gfp for selective photolabeling of proteins and cells. Science, 2002, 297: 1873–1877ADSCrossRefPubMedGoogle Scholar
  23. 23.
    Subach F V, Patterson G H, Manley S, et al. Photoactivatable mcherry for high-resolution two-color fluorescence microscopy. Nat Methods, 2009, 6: 153–159CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Subach F V, Patterson G H, Renz M, et al. Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptpalm of live cells. J Am Chem Soc, 2010, 132: 6481–6491CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ando R, Mizuno H, Miyawaki A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science, 2004, 306: 1370–1373ADSCrossRefPubMedGoogle Scholar
  26. 26.
    Chang H, Zhang M, Ji W, et al. A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications. Proc Natl Acad Sci USA, 2012, 109: 4455–4460ADSCrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Biteen J S, Thompson M A, Tselentis N K, et al. Super-resolution imaging in live caulobacter crescentus cells using photoswitchable eyfp. Nat Methods, 2008, 5: 947–949CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Henderson J N, Ai H W, Campbell R E, et al. Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc Natl Acad Sci USA, 2007, 104: 6672–6677ADSCrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Chudakov D M, Belousov V V, Zaraisky A G, et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol, 2003, 21: 191–194CrossRefPubMedGoogle Scholar
  30. 30.
    Shaner N C, Lin M Z, McKeown M R, et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods, 2008, 5: 545–551CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Stiel A C, Andresen M, Bock H, et al. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophys J, 2008, 95: 2989–2997CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Subach F V, Zhang L, Gadella T W, et al. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic fret. Chem Biol, 2010, 17: 745–755CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Mizuno H, Mal T K, Tong K I, et al. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol Cell, 2003, 12: 1051–1058CrossRefPubMedGoogle Scholar
  34. 34.
    Subach O M, Patterson G H, Ting L M, et al. A photoswitchable orange-to-far-red fluorescent protein, psmorange. Nat Methods, 2011, 8: 771–777CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Bogdanov A M, Mishin A S, Yampolsky I V, et al. Green fluorescent proteins are light-induced electron donors. Nat Chem Biol, 2009, 5: 459–461CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Wiedenmann J, Ivanchenko S, Oswald F, et al. Eosfp, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA, 2004, 101: 15905–15910ADSCrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    McKinney S A, Murphy C S, Hazelwood K L, et al. A bright and photostable photoconvertible fluorescent protein. Nat Methods, 2009, 6: 131–133CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Nienhaus G U, Nienhaus K, Holzle A, et al. Photoconvertible fluorescent protein eosfp: Biophysical properties and cell biology applications. Photochem Photobiol, 2006, 82: 351–358CrossRefPubMedGoogle Scholar
  39. 39.
    Wiedenmann J, Gayda S, Adam V, et al. From eosfp to mirisfp: Structure-based development of advanced photoactivatable marker proteins of the gfp-family. J Biophotonics, 2011, 4: 377–390CrossRefPubMedGoogle Scholar
  40. 40.
    Ando R, Hama H, Yamamoto-Hino M, et al. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci USA, 2002, 99: 12651–12656ADSCrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Habuchi S, Tsutsui H, Kochaniak A B, et al. Mkikgr, a monomeric photoswitchable fluorescent protein. PLoS One, 2008, 3: e3944ADSCrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Hoi H, Shaner N C, Davidson M W, et al. A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. J Mol Biol, 2010, 401: 776–791CrossRefPubMedGoogle Scholar
  43. 43.
    McEvoy A L, Hoi H, Bates M, et al. Mmaple: A photoconvertible fluorescent protein for use in multiple imaging modalities. PLoS One, 2012, 7: e51314ADSCrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Gurskaya N G, Verkhusha V V, Shcheglov A S, et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol, 2006, 24: 461–465CrossRefPubMedGoogle Scholar
  45. 45.
    Chudakov D M, Verkhusha V V, Staroverov D B, et al. Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol, 2004, 22: 1435–1439CrossRefPubMedGoogle Scholar
  46. 46.
    Fuchs J, Bohme S, Oswald F, et al. A photoactivatable marker protein for pulse-chase imaging with superresolution. Nat Methods, 2010, 7: 627–630CrossRefPubMedGoogle Scholar
  47. 47.
    Adam V, Moeyaert B, David C C, et al. Rational design of photoconvertible and biphotochromic fluorescent proteins for advanced microscopy applications. Chem Biol, 2011, 18: 1241–1251CrossRefPubMedGoogle Scholar
  48. 48.
    van de Linde S, Loschberger A, Klein T, et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc, 2011, 6: 991–1009CrossRefPubMedGoogle Scholar
  49. 49.
    Wombacher R, Heidbreder M, van de Linde S, et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods, 2010, 7: 717–719CrossRefPubMedGoogle Scholar
  50. 50.
    Jones S A, Shim S H, He J, et al. Fast, three-dimensional super-resolution imaging of live cells. Nat Methods, 2011, 8: 499–508CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Klein T, Loschberger A, Proppert S, et al. Live-cell dstorm with snap-tag fusion proteins. Nat Methods, 2011, 8: 7–9CrossRefPubMedGoogle Scholar
  52. 52.
    Ries J, Kaplan C, Platonova E, et al. A simple, versatile method for gfp-based super-resolution microscopy via nanobodies. Nat Methods, 2012, 9: 582–584CrossRefPubMedGoogle Scholar
  53. 53.
    Lelek M, Di Nunzio F, Henriques R, et al. Superresolution imaging of hiv in infected cells with flash-palm. Proc Natl Acad Sci USA, 2012, 109: 8564–8569ADSCrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Bock H, Geisler C, Wurm C A, et al. Two-color far-field fluorescence nanoscopy based on photoswitchable emitters. Appl Phys B, 2007, 88: 161–165ADSCrossRefGoogle Scholar
  55. 55.
    Bates M, Huang B, Dempsey G T, et al. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science, 2007, 317: 1749–1753ADSCrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Shroff H, Galbraith C G, Galbraith J A, et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci USA, 2007, 104: 20308–20313ADSCrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Huang B, Wang W Q, Bates M, et al. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science, 2008, 319: 810–813ADSCrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Juette M F, Gould T J, Lessard M D, et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods, 2008, 5: 527–529CrossRefPubMedGoogle Scholar
  59. 59.
    Pavani S R, Thompson M A, Biteen J S, et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc Natl Acad Sci USA, 2009, 106: 2995–2999ADSCrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Shtengel G, Galbraith J A, Galbraith C G, et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci USA, 2009, 106: 3125–3130ADSCrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Baddeley D, Crossman D, Rossberger S, et al. 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. PLoS One, 2011, 6: e20645ADSCrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Xu K, Babcock H P, Zhuang X. Dual-objective storm reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods, 2012, 9: 185–188CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Axelrod D, Burghardt T P, Thompson N L. Total internal reflection fluorescence. Annu Rev Biophys Bioeng, 1984, 13: 247–268CrossRefPubMedGoogle Scholar
  64. 64.
    Tokunaga M, Imamoto N, Sakata-Sogawa K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods, 2008, 5: 159–161CrossRefPubMedGoogle Scholar
  65. 65.
    York A G, Ghitani A, Vaziri A, et al. Confined activation and subdiffractive localization enables whole-cell palm with genetically expressed probes. Nat Methods, 2011, 8: 327–333CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Cella Z F, Lavagnino Z, Perrone D M, et al. Live-cell 3D super-resolution imaging in thick biological samples. Nat Methods, 2011, 8: 1047–1049CrossRefGoogle Scholar
  67. 67.
    Truong T V, Supatto W, Koos D S, et al. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat Methods, 2011, 8: 757–760CrossRefPubMedGoogle Scholar
  68. 68.
    Shim S H, Xia C, Zhong G, et al. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc Natl Acad Sci USA, 2012, 109: 13978–13983ADSCrossRefPubMedPubMedCentralGoogle Scholar
  69. 69.
    Abraham A V, Ram S, Chao J, et al. Quantitative study of single molecule location estimation techniques. Opt Express, 2009, 17: 23352–23373ADSCrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Smith C S, Joseph N, Rieger B, et al. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods, 2010, 7: 373–375CrossRefPubMedPubMedCentralGoogle Scholar
  71. 71.
    Hedde P N, Fuchs J, Oswald F, et al. Online image analysis software for photoactivation localization microscopy. Nat Methods, 2009, 6: 689–690CrossRefPubMedGoogle Scholar
  72. 72.
    Parthasarathy R. Rapid, accurate particle tracking by calculation of radial symmetry centers. Nat Methods, 2012, 9: 724–726CrossRefPubMedGoogle Scholar
  73. 73.
    Holden S J, Uphoff S, Kapanidis A N. Daostorm: An algorithm for high-density super-resolution microscopy. Nat Methods, 2011, 8: 279–280CrossRefPubMedGoogle Scholar
  74. 74.
    Zhu L, Zhang W, Elnatan D, et al. Faster storm using compressed sensing. Nat Methods, 2012, 9: 721–723CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Mukamel E A, Babcock H, Zhuang X W. Statistical deconvolution for superresolution fluorescence microscopy. Biophys J, 2012, 102: 2391–2400CrossRefPubMedPubMedCentralGoogle Scholar
  76. 76.
    Huang F, Schwartz S L, Byars J M, et al. Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomedical Optics Express, 2011, 2: 1377–1393CrossRefPubMedPubMedCentralGoogle Scholar
  77. 77.
    Dertinger T, Colyer R, Iyer G, et al. Fast, background-free, 3D super-resolution optical fluctuation imaging (sofi). Proc Natl Acad Sci USA, 2009, 106: 22287–22292ADSCrossRefPubMedPubMedCentralGoogle Scholar
  78. 78.
    Burnette D T, Sengupta P, Dai Y, et al. Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules. Proc Natl Acad Sci USA, 2011, 108: 21081–21086ADSCrossRefPubMedPubMedCentralGoogle Scholar
  79. 79.
    Simonson P D, Rothenberg E, Selvin P R. Single-molecule-based super-resolution images in the presence of multiple fluorophores. Nano Lett, 2011, 11: 5090–5096ADSCrossRefPubMedPubMedCentralGoogle Scholar
  80. 80.
    Cox S, Rosten E, Monypenny J, et al. Bayesian localization microscopy reveals nanoscale podosome dynamics. Nat Methods, 2012, 9: 195–200CrossRefGoogle Scholar
  81. 81.
    Lee S H, Baday M, Tjioe M, et al. Using fixed fiduciary markers for stage drift correction. Opt Express, 2012, 20: 12177–12183ADSCrossRefPubMedPubMedCentralGoogle Scholar
  82. 82.
    Geisler C, Hotz T, Schonle A, et al. Drift estimation for single marker switching based imaging schemes. Opt Express, 2012, 20: 7274–7289ADSCrossRefPubMedGoogle Scholar
  83. 83.
    Mlodzianoski M J, Schreiner J M, Callahan S P, et al. Sample drift correction in 3D fluorescence photoactivation localization microscopy. Opt Express, 2011, 19: 15009–15019ADSCrossRefPubMedGoogle Scholar
  84. 84.
    Vaughan J C, Jia S, Zhuang X. Ultrabright photoactivatable fluorophores created by reductive caging. Nat Methods, 2012, 9: 1181–1184CrossRefPubMedPubMedCentralGoogle Scholar
  85. 85.
    van Rijnsoever C, Oorschot V, Klumperman J. Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods, 2008, 5: 973–980CrossRefPubMedGoogle Scholar
  86. 86.
    Watanabe S, Punge A, Hollopeter G, et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat Methods, 2011, 8: 80–84CrossRefPubMedGoogle Scholar
  87. 87.
    Kopek B G, Shtengel G, Xu C S, et al. Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes. Proc Natl Acad Sci USA, 2012, 109: 6136–6141ADSCrossRefPubMedPubMedCentralGoogle Scholar
  88. 88.
    Henriques R, Lelek M, Fornasiero E F, et al. Quickpalm: 3D real-time photoactivation nanoscopy image processing in image. Nat Methods, 2010, 7: 339–340CrossRefPubMedGoogle Scholar
  89. 89.
    Wolter S, Loschberger A, Holm T, et al. Rapidstorm: Accurate, fast open-source software for localization microscopy. Nat Methods, 2012, 9: 1040–1041CrossRefPubMedGoogle Scholar
  90. 90.
    Ji N, Milkie D E, Betzig E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat Methods, 2010, 7: 141–147CrossRefPubMedGoogle Scholar
  91. 91.
    Stallinga S, Rieger B. Accuracy of the gaussian point spread function model in 2D localization microscopy. Opt Express, 2010, 18: 24461–24476ADSCrossRefPubMedGoogle Scholar
  92. 92.
    Engelhardt J, Keller J, Hoyer P, et al. Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy. Nano Lett, 2011, 11: 209–213ADSCrossRefPubMedGoogle Scholar

Copyright information

© The Author(s) 2013

Authors and Affiliations

  1. 1.School of Life Science and TechnologyHuazhong University of Science and TechnologyWuhanChina
  2. 2.School of Life ScienceUniversity of Science and Technology of ChinaHefeiChina
  3. 3.Institute of BiophysicsChinese Academy of SciencesBeijingChina

Personalised recommendations