European Biophysics Journal

, Volume 38, Issue 6, pp 807–812 | Cite as

Structured illumination microscopy of a living cell

  • Liisa M. Hirvonen
  • Kai Wicker
  • Ondrej Mandula
  • Rainer Heintzmann
Original Paper


Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells.


Structured illumination microscopy Live-cell imaging Resolution improvement 



This work was funded by the Medical Research Council, Carl Zeiss MicroImaging GmbH and the International Agency for Atomic Energy.


  1. Amodaj N, Stuurman N (2006–2009) μManager.
  2. Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373PubMedCrossRefGoogle Scholar
  3. Bailey B, Farkas DL, Taylor DL, Lanni F (1993) Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature 366:44–48PubMedCrossRefGoogle Scholar
  4. Bates M, Blosser TR, Zhuang X (2005) Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett 94:101–108CrossRefGoogle Scholar
  5. 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 313:1642–1645PubMedCrossRefGoogle Scholar
  6. Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO, Lührmann R, Jahn R, Eggeling C, Hell SW (2006) Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci USA 103(31):11440–11445PubMedCrossRefGoogle Scholar
  7. Failla AV, Spoeri U, Albrecht B, Kroll A, Cremer C (2002) Nanosizing fluorescent objects by spatially modulated illumination microscopy. Appl Opt 41(34):7275–7283PubMedCrossRefGoogle Scholar
  8. Geisler C, Schönle A, von Middendorff C, Bock H, Eggeling C, Egner A, Hell SW (2007) Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching. Appl Phys A 88:223–226CrossRefGoogle Scholar
  9. Gustafsson MGL (2000) Surpassing the lateral resolution limit by a factor of two using stuctured illumination microscopy. J Microsc 198:82–87PubMedCrossRefGoogle Scholar
  10. Gustafsson MGL (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102(37):13081–13086PubMedCrossRefGoogle Scholar
  11. Gustafsson MGL, Shao L, Carlton PM, Wang CJR, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94:4957–4970PubMedCrossRefGoogle Scholar
  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 127:3801–3806PubMedCrossRefGoogle Scholar
  13. Heintzmann R (2003) Saturated patterned excitation microscopy with two-dimensional excitation patterns. Micron 34:283–291PubMedCrossRefGoogle Scholar
  14. Heintzmann R, Cremer C (1998) Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc SPIE 3568:185–195CrossRefGoogle Scholar
  15. Heintzmann R, Jovin TM, Cremer C (2002) Saturated patterned excitation microscopy—a concept for optical resolution improvement. J Opt Soc Am A 19(8):1599–1609CrossRefGoogle Scholar
  16. Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782CrossRefGoogle Scholar
  17. Hess ST, Girirajan TPK, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272PubMedCrossRefGoogle Scholar
  18. Hirvonen L, Mandula O, Wicker K, Heintzmann R (2008) Structured illumination microscopy using photoswitchable fluorescent proteins. Proc SPIE 6861:68610LCrossRefGoogle Scholar
  19. Huang B, Jones SA, Brandenburg B, Zhuang X (2008) Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5(12):1047–1052PubMedCrossRefGoogle Scholar
  20. 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):527–529PubMedCrossRefGoogle Scholar
  21. Kner P, Chhun BB, Griffis E, Winoto L, Gustafsson MGL (2009) Super-resolution video microscopy of live cells by structured illumination. Nat Methods 6(5):339–342PubMedCrossRefGoogle Scholar
  22. Lemmer P, Gunkel M, Baddeley D, Kaufmann R, Urich A, Weiland Y, Reymann J, Müller P, Hausmann M, Cremer C (2008) SPDM: light microscopy with single-molecule resolution at the nanoscale. Appl Phys B93:1–12Google Scholar
  23. Lidke KA, Rieger B, Jovin TM, Heintzmann R (2005) Superresolution by localization of quantum dots using blinking statistics. Opt Exp 13(18):7052–7062CrossRefGoogle Scholar
  24. Lukyanov KA, Fradkov AF, Gurskaya NG, Matz MV, Labas YA, Savitsky AP, Markelov ML, Zaraisky AG, Zhao XN, Fang Y, Tan W, Lukyanov SA (2000) Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem 275:25879–25882PubMedCrossRefGoogle Scholar
  25. Mandula O (2008) Patterned excitation microscopy. Master’s Thesis, Charles University, PragueGoogle Scholar
  26. Niemeyer P (2008) Beanshell.
  27. Pham TQ, Bezuijen M, van Vliet LJ, Schutte K, Luengo Hendriks CL (2005) Performance of optimal registration estimators. In: Rahman Z, Schowengerdt RA, Reichenbach SE (eds) Visual information processing XIV, Orlando, FL, USA. Proceedings of SPIE, vol. 5817, pp 133–144.Google Scholar
  28. Rittweger E, Han KY, Irvine SE, Eggeling C, Hell SW (2009) STED microscopy reveals crystal colour centres with nanometric resolution. Nat Photonics 3:144–147CrossRefGoogle Scholar
  29. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795PubMedCrossRefGoogle Scholar
  30. Sabanayagam CR, Eid JS, Meller A (2005) Long time scale blinking kinetics of cyanine fluorophores conjugated to DNA and its effect on Förster resonance energy transfer. J Chem Phys 123:224–708CrossRefGoogle Scholar
  31. Schrader M, Meinecke F, Bahlmann K, Kroug M, Cremer C, Soini E, Hell SW (1995) Monitoring the excited state of a fluorophore in a microscope by stimulated emission. Bioimaging 3:147–153CrossRefGoogle Scholar
  32. Shao L, Isaac B, Uzawa S, Agard DA, Sedat JW, Gustafsson MGL (2008) I5S: wide-field light microscopy with 100-nm-scale resolution in three dimensions. Biophys J 94:4971–4983PubMedCrossRefGoogle Scholar
  33. Shroff H, Galbraith CG, Galbraith JA, Betzig E (2008) Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5(5):417–423PubMedCrossRefGoogle Scholar
  34. Stuurman N, Amodaj N, Vale R (2007) μManager: open source software for light microscope imaging. Microsc Today 15:42–43Google Scholar

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • Liisa M. Hirvonen
    • 1
  • Kai Wicker
    • 1
  • Ondrej Mandula
    • 2
  • Rainer Heintzmann
    • 1
  1. 1.Randall Division of Cell and Molecular BiophysicsKing’s College LondonLondonUK
  2. 2.Faculty of Mathematics and PhysicsCharles UniversityPraha 2Czech Republic

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