Photoswitchable Fluorescent Proteins for Superresolution Fluorescence Microscopy Circumventing the Diffraction Limit of Light

  • Susana Rocha
  • Herlinde De Keersmaecker
  • Hiroshi Uji-i
  • Johan Hofkens
  • Hideaki Mizuno
Part of the Methods in Molecular Biology book series (MIMB, volume 1076)


In the last two decades, fluorescent proteins became an indispensable tool to noninvasively label a protein in living cells. The discovery of photoswitchable fluorescent proteins expanded the applications of the fluorescent proteins to techniques such as molecular tracking and highlighting on a microscope. Recently, a new microscopic modality to achieve a superresolution circumventing the diffraction limit of light (photoactivated localization microscopy, PALM) has been developed based on the photoswitchable fluorescent proteins. Here we introduce a basic protocol of PALM through the visualization of actin bundles with superresolution.

Key words

Actin bundles Diffraction limit Fluorescent protein Photoswitchable fluorescent protein Superresolution microscopy PALM 



We thank Dr. A. Miyawaki and Mr. S. Karasawa (Brain Science Institute, RIKEN, Japan) for hKikGR-β-actin/pMC1.


  1. 1.
    Prasher DC, Eckenrode VK, Ward WW et al (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229–233PubMedCrossRefGoogle Scholar
  2. 2.
    Chalfie M, Tu Y, Euskirchen G, Ward W et al (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805PubMedCrossRefGoogle Scholar
  3. 3.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544PubMedCrossRefGoogle Scholar
  4. 4.
    Tsien RY (2005) Building and breeding molecules to spy on cells and tumors. FEBS Lett 579:927–932PubMedCrossRefGoogle Scholar
  5. 5.
    Miyawaki A (2005) Innovations in the imaging of brain functions using fluorescent proteins. Neuron 48:189–199PubMedCrossRefGoogle Scholar
  6. 6.
    Newman RH, Fosbrink MD, Zhang J (2011) Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111:3614–3666PubMedCrossRefGoogle Scholar
  7. 7.
    Patterson GH (2011) Highlights of the optical highlighter fluorescent proteins. J Microsc 243:1–7PubMedCrossRefGoogle Scholar
  8. 8.
    Ando R, Hama H, Yamamoto-Hino M et al (2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci 99:12651–12656PubMedCrossRefGoogle Scholar
  9. 9.
    Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373PubMedCrossRefGoogle Scholar
  10. 10.
    Mizuno H, Mal TK, Tong KI et al (2003) Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol Cell 12:1051–1058PubMedCrossRefGoogle Scholar
  11. 11.
    Sato T, Takahoko M, Okamoto H (2006) HuC:Kaede, a useful tool to label neural morphologies in networks in vivo. Genesis 44:136–142PubMedCrossRefGoogle Scholar
  12. 12.
    Adam V, Mizuno H, Grichine A et al (2010) Data storage based on photochromic and photoconvertible fluorescent proteins. J Biotechnol 149:289–298PubMedCrossRefGoogle Scholar
  13. 13.
    Grotjohann T, Testa I, Leutenegger M et al (2011) Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478:204–208PubMedCrossRefGoogle Scholar
  14. 14.
    Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877PubMedCrossRefGoogle Scholar
  15. 15.
    Henderson JN, Gepshtein R, Heenan JR et al (2009) Structure and mechanism of the photoactivatable green fluorescent protein. J Am Chem Soc 131:4176–4177PubMedCrossRefGoogle Scholar
  16. 16.
    Verkhusha VV, Sorkin A (2005) Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem Biol 12:279–285PubMedCrossRefGoogle Scholar
  17. 17.
    Subach FV, Patterson GH, Manley S et al (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6:153–159PubMedCrossRefGoogle Scholar
  18. 18.
    Subach FV, Patterson GH, Renz M et al (2010) Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. J Am Chem Soc 132:6481–6491PubMedCrossRefGoogle Scholar
  19. 19.
    Wiedenmann J, Ivanchenko S, Oswald F et al (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA 101:15905–15910PubMedCrossRefGoogle Scholar
  20. 20.
    Tsutsui H, Karasawa S, Shimizu H et al (2005) Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep 6:233–238PubMedCrossRefGoogle Scholar
  21. 21.
    Gurskaya NG, Verkhusha VV, Shcheglov AS et al (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24:461–465PubMedCrossRefGoogle Scholar
  22. 22.
    Habuchi S, Tsutsui H, Kochaniak AB et al (2008) mKikGR, a monomeric photoswitchable fluorescent protein. PLoS One 3:e3944PubMedCrossRefGoogle Scholar
  23. 23.
    McKinney SA, Murphy CS, Hazelwood KL et al (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133PubMedCrossRefGoogle Scholar
  24. 24.
    Zhang M, Chang H, Zhang Y et al (2012) Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods 9:727–729PubMedCrossRefGoogle Scholar
  25. 25.
    Chudakov DM, Verkhusha VV, Staroverov DB et al (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22:1435–1439PubMedCrossRefGoogle Scholar
  26. 26.
    Andresen M, Stiel AC, Trowitzsch S et al (2007) Structural basis for reversible photoswitching in Dronpa. Proc Natl Acad Sci USA 104:13005–13009PubMedCrossRefGoogle Scholar
  27. 27.
    Mizuno H, Mal TK, Wälchli M et al (2010) Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination. J Biomol NMR 48:237–246PubMedCrossRefGoogle Scholar
  28. 28.
    Stiel AC, Trowitzsch S, Weber G et al (2007) 1.8 Å bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem J 402:35–42PubMedCrossRefGoogle Scholar
  29. 29.
    Ando R, Flors C, Mizuno H et al (2007) Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants. Biophys J 92:L97–L99PubMedCrossRefGoogle Scholar
  30. 30.
    Andresen M, Stiel AC, Fölling J et al (2008) Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26:1035–1040PubMedCrossRefGoogle Scholar
  31. 31.
    Stiel AC, Andresen M, Bock H et al (2008) Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophys J 95:2989–2997PubMedCrossRefGoogle Scholar
  32. 32.
    Subach FV, Zhang L, Gadella TW et al (2010) Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET. Chem Biol 17:745–755PubMedCrossRefGoogle Scholar
  33. 33.
    Brakemann T, Stiel AC, Weber G et al (2011) A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat Biotechnol 29:942PubMedCrossRefGoogle Scholar
  34. 34.
    Adam V, Lelimousin M, Boehme S et al (2008) Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. Proc Natl Acad Sci USA 105:18343–18348PubMedCrossRefGoogle Scholar
  35. 35.
    Fuchs J, Böhme S, Oswald F et al (2010) A photoactivatable marker protein for pulse-chase imaging with superresolution. Nat Methods 7:627–630PubMedCrossRefGoogle Scholar
  36. 36.
    Adam V, Moeyaert B, David CC et al (2011) Rational design of photoconvertible and biphotochromic fluorescent proteins for advanced microscopy applications. Chem Biol 18:1241–1251PubMedCrossRefGoogle Scholar
  37. 37.
    Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645PubMedCrossRefGoogle Scholar
  38. 38.
    Hess ST, Girirajan T, Mason M (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272PubMedCrossRefGoogle Scholar
  39. 39.
    Mizuno H, Dedecker P, Ando R et al (2010) Higher resolution in localization microscopy by slower switching of a photochromic protein. Photochem Photobiol Sci 9:239–248PubMedCrossRefGoogle Scholar
  40. 40.
    Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28PubMedCrossRefGoogle Scholar
  41. 41.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, NYGoogle Scholar
  42. 42.
    van de Linde S, Wolter S, Heilemann M et al (2010) The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. J Biotechnol 149:260–266PubMedCrossRefGoogle Scholar
  43. 43.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Susana Rocha
    • 1
  • Herlinde De Keersmaecker
    • 1
  • Hiroshi Uji-i
    • 1
  • Johan Hofkens
    • 1
  • Hideaki Mizuno
    • 1
  1. 1.Department of ChemistryKU LeuvenLeuvenBelgium

Personalised recommendations