Sub-Diffraction-Limit Imaging with Stochastic Optical Reconstruction Microscopy

  • Mark Bates
  • Bo Huang
  • Michael J. Rust
  • Graham T. Dempsey
  • Wenqin Wang
  • Xiaowei Zhuang
Part of the Springer Series in Chemical Physics book series (CHEMICAL, volume 96)


Light microscopy is a widely used imaging method in biomedical research. However, the resolution of conventional optical microcopy is limited by the diffraction of light, making structures smaller than 200 nm difficult to resolve. To overcome this limit, we have developed a new form of fluorescence microscopy - Stochastic Optical Reconstruction Microscopy (STORM). STORM makes use of single-molecule imaging methods and photo-switchable fluorescent probes to temporally separate the otherwise spatially overlapping images of individual molecules. An STORM image is acquired over a number of imaging cycles, and in each cycle only a subset of the fluorescent labels is switched on such that each of the active fluorophores is optically resolvable from the rest. This allows the position of these fluorophores to be determined with nanometer accuracy. Over the course of many such cycles, the positions of numerous fluorophores are determined and used to construct a super-resolution image. Using this method, we have demonstrated multi-color, three-dimensional (3D) imaging of biomolecules and cells with ∼ 20 nm lateral and ∼ 50 nm axial resolutions. In principle, the resolution of this technique can reach the molecular scale.


Localization Accuracy Centroid Position Superresolution Imaging Imaging Cycle Stochastic Optical Reconstruction Microscopy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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This work is supported by in part by the NIH (to X.Z.). X.Z. is a Howard Hughes Medical Institute Investigator.


  1. 1.
    S.W. Hell, J. Wichmann, Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994)CrossRefADSGoogle Scholar
  2. 2.
    S.W. Hell, Far-field optical nanoscopy. Science 316, 1153–1158 (2007)Google Scholar
  3. 3.
    M.G.L. Gustafsson, Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. U S A 102, 13081–13086 (2005)CrossRefADSGoogle Scholar
  4. 4.
    M.J. Rust, M. Bates, X. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Meth. 3, 793–795 (2006)CrossRefGoogle Scholar
  5. 5.
    E. Betzig, G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, H.F. Hess, Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006)CrossRefADSGoogle Scholar
  6. 6.
    S.T. Hess, T.P. Girirajan, M.D. Mason, Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006)CrossRefADSGoogle Scholar
  7. 7.
    W.E. Moerner, M. Orrit, Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999)CrossRefADSGoogle Scholar
  8. 8.
    J. Gelles, B.J. Schnapp, M.P. Sheetz, Tracking kinesin-driven movements with nanometre-scale precision. Nature 331, 450–453 (1988)CrossRefADSGoogle Scholar
  9. 9.
    R.N. Ghosh, W.W. Webb, Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules. Biophys. J. 66, 1301–1318 (1994)CrossRefADSGoogle Scholar
  10. 10.
    R.E. Thompson, D.R. Larson, W.W. Webb, Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002)CrossRefGoogle Scholar
  11. 11.
    A. Yildiz, J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, P.R. Selvin, Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003)Google Scholar
  12. 12.
    A.M. van Oijen, J. Kohler, J. Schmidt, M. Muller, G.J. Brakenhoff, 3-Dimensional super-resolution by spectrally selective imaging. Chem. Phys. Lett. 292, 183–187 (1998)CrossRefGoogle Scholar
  13. 13.
    T.D. Lacoste, X. Michalet, F. Pinaud, D.S. Chemla, A.P. Alivisatos, S. Weiss, Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc. Natl. Acad. Sci. U S A 97, 9461–9466 (2000)CrossRefADSGoogle Scholar
  14. 14.
    L.S. Churchman, Z. Okten, R.S. Rock, J.F. Dawson, J.A. Spudich, Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc. Natl. Acad. Sci. U S A 102, 1419–1423 (2005)CrossRefADSGoogle Scholar
  15. 15.
    M.P. Gordon, T. Ha, P.R. Selvin, Single-molecule high-resolution imaging with photobleaching. Proc. Natl. Acad. Sci. U S A 101, 6462–6465 (2004)CrossRefADSGoogle Scholar
  16. 16.
    X. Qu, D. Wu, L. Mets, N.F. Scherer, Nanometer-localized multiple single-molecule fluorescence microscopy. Proc. Natl. Acad. Sci. U S A 101, 11298–11303 (2004)CrossRefADSGoogle Scholar
  17. 17.
    K. Lidke, B. Rieger, T. Jovin, R. Heintzmann, Superresolution by localization of quantum dots using blinking statistics. Opt. Exp. 13, 7052–7062 (2005)CrossRefADSGoogle Scholar
  18. 18.
    M. Bates, T.R. Blosser, X. Zhuang, Short-range spectroscopic ruler based on a single-molecule optical switch. Phys. Rev. Lett. 94, 108101 (2005)Google Scholar
  19. 19.
    M. Bates, B. Huang, G.T. Dempsey, X. Zhuang, Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007)CrossRefADSGoogle Scholar
  20. 20.
    M. Heilemann, E. Margeat, R. Kasper, M. Sauer, P. Tinnefeld, Carbocyanine dyes as efficient reversible single-molecule optical switch. J. Am. Chem. Soc. 127, 3801–3806 (2005)CrossRefGoogle Scholar
  21. 21.
    S. Hohng, C. Joo, T. Ha, Single-molecule three-color FRET. Biophys. J. 87, 1328–1337 (2004)Google Scholar
  22. 22.
    J. Folling, V. Belov, R. Kunetsky, R. Medda, A. Schonle, A. Egner, C. Eggeling, M. Bossi, S.W. Hell, Photochromic rhodamines provide nanoscopy with optical sectioning. Angew. Chem. Int. Ed. Engl. 46, 6266–6270 (2007)CrossRefGoogle Scholar
  23. 23.
    G.H. Patterson, J. Lippincott-Schwartz, A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002)CrossRefADSGoogle Scholar
  24. 24.
    R. Ando, H. Hama, M. Yamamoto-Hino, H. Mizuno, A. Miyawaki, An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. U S A 99, 12651–12656 (2002)CrossRefADSGoogle Scholar
  25. 25.
    J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Rocker, A. Salih, K.D. Spindler, G.U. Nienhaus, EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc. Natl. Acad. Sci. U S A 101, 15905–15910 (2004)CrossRefADSGoogle Scholar
  26. 26.
    H. Tsutsui, S. Karasawa, H. Shimizu, N. Nukina, A. Miyawaki, Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6, 233–238 (2005)CrossRefGoogle Scholar
  27. 27.
    S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, J. Hofkens, Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc. Natl. Acad. Sci. U S A 102, 9511–9516 (2005)CrossRefADSGoogle Scholar
  28. 28.
    R. Ando, C. Flors, H. Mizuno, J. Hofkens, A. Miyawaki, Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants. Biophys. J. 92, L97–99 (2007)CrossRefGoogle Scholar
  29. 29.
    A.C. Stiel, S. Trowitzsch, G. Weber, M. Andresen, C. Eggeling, S.W. Hell, S. Jakobs, M.C. Wahl, 1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem. J. 402, 35–42 (2007)Google Scholar
  30. 30.
    D.M. Chudakov, V.V. Verkhusha, D.B. Staroverov, E.A. Souslova, S. Lukyanov, K.A. Lukyanov, Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22, 1435–1439 (2004)CrossRefGoogle Scholar
  31. 31.
    C. Bustamante, J.F. Marko, E.D. Siggia, S. Smith, Entropic elasticity of lambda-phage DNA. Science 265, 1599–1600 (1994)Google Scholar
  32. 32.
    A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A.C. Stiel, S. Jakobs, C. Eggeling, et al., Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007)CrossRefADSGoogle Scholar
  33. 33.
    J.E. Heuser, R.G.W. Anderson, Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. 108, 389–400 (1989)CrossRefGoogle Scholar
  34. 34.
    H. Shroff, C.G. Galbraith, J.A. Galbraith, H. White, J. Gillette, S. Olenych, M.W. Davidson, E. Betzig, Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc. Natl. Acad. Sci. U S A 104, 20308–20313 (2007)CrossRefADSGoogle Scholar
  35. 35.
    H. Bock, C. Geisler, C.A. Wurm, C. Von Middendorff, S. Jakobs, A. Schonle, A. Egner, S.W. Hell, C. Eggeling, Two-color far-field fluorescence nanoscopy based on photoswitchable emitters. Appl. Phys. B 88, 161–165 (2007)CrossRefADSGoogle Scholar
  36. 36.
    E. Toprak, H. Balci, B.H. Blehm, P.R. Selvin, Three-dimensional particle tracking via bifocal imaging. Nano Lett. 7, 2043–2045 (2007)CrossRefADSGoogle Scholar
  37. 37.
    H.P. Kao, A.S. Verkman, Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys. J. 67, 1291–1300 (1994)CrossRefADSGoogle Scholar
  38. 38.
    L. Holtzer, T. Meckel, T. Schmidt, Nanometric three-dimensional tracking of individual quantum dots in cells. Appl. Phys. Lett. 90, 053902 (2007)Google Scholar
  39. 39.
    B. Huang, W. Wang, M. Bates, X. Zhuang, Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008)CrossRefADSGoogle Scholar
  40. 40.
    M.F. Juette, T.J. Gould, M.D. Lessard, M.J. Moldzianoski, B.S. Nagpure, B.T. Bennett, S.T. Hess, J. Bewersdorf, Three-dimernsional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Meth. 5, 527–529 (2008)CrossRefGoogle Scholar
  41. 41.
    R. Schmidt, C.A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, S.W. Hell, Spherical nanosized focal spot unravels the interior of cells. Nat. Meth. 5, 539–544 (2008)CrossRefGoogle Scholar
  42. 42.
    L. Schermelleh, P.M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M.C. Cardoso, D.A. Agard, M.G.L. Gustafsson, et al., Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008)CrossRefADSGoogle Scholar
  43. 43.
    H. Shroff, C.G. Galbraith, J.A. Galbraith, E. Betzig, Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Meth. 5, 417–423 (2008)CrossRefGoogle Scholar
  44. 44.
    B.A. Griffin, S.R. Adams, R.Y. Tsien, Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998)CrossRefADSGoogle Scholar
  45. 45.
    E.G. Guignet, R. Hovius, H. Vogel, Reversible site-selective labeling of membrane proteins in live cells. Nat. Biotechnol. 22, 440–444 (2004)CrossRefGoogle Scholar
  46. 46.
    I. Chen, M. Howarth, W. Lin, A. Ting, Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Meth. 2, 99–104 (2005)CrossRefGoogle Scholar
  47. 47.
    M. Fernandez-Suarez, H. Baruah, L. Martinez-Hernandez, K.T. Xie, J.M. Baskin, C.R. Bertozzi, A.Y. Ting, Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat. Biotechnol. 25, 1483–1487 (2007)CrossRefGoogle Scholar
  48. 48.
    M.W. Popp, J.M. Antos, G.M. Grotenbreg, E. Spooner, H.L. Ploegh, Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 3, 707–708 (2007)CrossRefGoogle Scholar
  49. 49.
    B. Huang, S.A. Jones, B. Brandenberg, X. Zhuang, Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Meth. 5, 1047–1082 (2008)Google Scholar
  50. 50.
    Pavani et al, Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit by using a double helical point spread function. Proc. Natl. Acad. Sci. USA 106, 2995–2999 (2009)Google Scholar
  51. 51.
    Shetengel et al, Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009)Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Mark Bates
    • 1
  • Bo Huang
    • 2
    • 3
  • Michael J. Rust
    • 4
  • Graham T. Dempsey
    • 5
  • Wenqin Wang
    • 4
  • Xiaowei Zhuang
    • 6
  1. 1.School of Engineering and Applied SciencesCambridgeUSA
  2. 2.Department of Chemistry and Chemical BiologyCornell UniversityIthacaUSA
  3. 3.Howard Hughes Medical Institute Harvard UniversityCambridgeUSA
  4. 4.Department of PhysicsHarvard UniversityCambridgeUSA
  5. 5.Program in BiophysicsHarvard UniversityCambridgeUSA
  6. 6.Department of Chemistry and Chemical BiologyHoward Hughes Medical Institute Harvard UniversityCambridgeUSA

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