Advertisement

Localization-Based Super-Resolution Imaging of Cellular Structures

  • Pakorn Kanchanawong
  • Clare M. Waterman
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1046)

Abstract

Fluorescence microscopy allows direct visualization of fluorescently tagged proteins within cells. However, the spatial resolution of conventional fluorescence microscopes is limited by diffraction to ~250 nm, prompting the development of super-resolution microscopy which offers resolution approaching the scale of single proteins, i.e., ~20 nm. Here, we describe protocols for single molecule localization-based super-resolution imaging, using focal adhesion proteins as an example and employing either photoswitchable fluorophores or photoactivatable fluorescent proteins. These protocols should also be easily adaptable to imaging a broad array of macromolecular assemblies in cells whose components can be fluorescently tagged and assemble into high density structures.

Key words

Super-resolution microscopy Focal adhesions Localization microscopy TIRF PALM Single molecules Photoswitchable fluorophores Photoactivatable fluorescent proteins 

Notes

Acknowledgments

PK is supported by the Singapore National Research Foundation under the NRF Fellowship (NRFF-2011-04). CMW is supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health. We thank Harald Hess and Gleb Shtengel (Howard Hughes Medical Institute, Janelia Farm Research Campus), and Michael Davidson (The Florida State University) for advice, equipment, reagents, and collaboration related to this work.

References

  1. 1.
    Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61:345–367PubMedCrossRefGoogle Scholar
  2. 2.
    Galbraith CG, Galbraith JA (2011) Super-resolution microscopy at a glance. J Cell Sci 124:1607–1611PubMedCrossRefGoogle Scholar
  3. 3.
    Fernández-Suárez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9:929–943PubMedCrossRefGoogle Scholar
  4. 4.
    Heintzmann R, Gustafsson MGL (2009) Subdiffraction resolution in continuous samples. Nat Photonics 3:362–364CrossRefGoogle Scholar
  5. 5.
    Huang B, Bates M, Zhuang (2009) Super-resolution fluorescence microscopy. Annu Rev Biochem 78:993–1016Google Scholar
  6. 6.
    Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440:935–939PubMedCrossRefGoogle Scholar
  7. 7.
    Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci USA 97: 8206–8210PubMedCrossRefGoogle Scholar
  8. 8.
    Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82–87PubMedCrossRefGoogle Scholar
  9. 9.
    Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642–1645PubMedCrossRefGoogle Scholar
  10. 10.
    Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272PubMedCrossRefGoogle Scholar
  11. 11.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795PubMedCrossRefGoogle Scholar
  12. 12.
    Fölling J, Bossi M, Bock H et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5:943–945PubMedCrossRefGoogle Scholar
  13. 13.
    Shtengel G, Galbraith JA, Galbraith CG et al (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci USA 106:3125–3130PubMedCrossRefGoogle Scholar
  14. 14.
    Shroff H, Galbraith CG, Galbraith JA, Betzig E (2008) Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5:417–423PubMedCrossRefGoogle Scholar
  15. 15.
    Shroff H, Galbraith CG, Galbraith JA et al (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci USA 104:20308–20313PubMedCrossRefGoogle Scholar
  16. 16.
    Kanchanawong P, Shtengel G, Pasapera AM et al (2010) Nanoscale architecture of integrin-based cell adhesions. Nature 468:580–584PubMedCrossRefGoogle Scholar
  17. 17.
    Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388:355–358PubMedCrossRefGoogle Scholar
  18. 18.
    Levitus M, Ranjit S (2011) Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments. Q Rev Biophys 44:123–151PubMedCrossRefGoogle Scholar
  19. 19.
    Moerner WE (2007) New directions in single-molecule imaging and analysis. Proc Natl Acad Sci USA 104:12596–12602PubMedCrossRefGoogle Scholar
  20. 20.
    Shannon C (1949) Communication in the presence of noise. Proc IRE 37:10–21CrossRefGoogle Scholar
  21. 21.
    Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297: 1873–1877PubMedCrossRefGoogle Scholar
  22. 22.
    Subach FV, Patterson GH, Manley S, Gillette JM, Lippincott-Schwartz J, Verkhusha VV (2009) Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat Methods 6:153–159PubMedCrossRefGoogle Scholar
  23. 23.
    Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19:555–565PubMedCrossRefGoogle Scholar
  24. 24.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783PubMedCrossRefGoogle Scholar
  25. 25.
    Mortensen KI, Churchman LS, Spudich JA, Flyvbjerg H (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Methods 7: 377–381PubMedCrossRefGoogle Scholar
  26. 26.
    Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8:1027–1036PubMedCrossRefGoogle Scholar
  27. 27.
    Vogelsang J, Kasper R, Steinhauer C et al (2008) A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl 47: 5465–5469PubMedCrossRefGoogle Scholar
  28. 28.
    Aitken CE, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94:1826–1835PubMedCrossRefGoogle Scholar
  29. 29.
    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 7:339–340PubMedCrossRefGoogle Scholar
  30. 30.
    Wolter S, Endesfelder U, van de Linde S, Heilemann M, Sauer M (2011) Measuring localization performance of super-resolution algorithms on very active samples. Opt Express 19:7020–7033PubMedCrossRefGoogle Scholar
  31. 31.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682PubMedCrossRefGoogle Scholar
  32. 32.
    Miller JC, Tan S, Qiao G et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148PubMedCrossRefGoogle Scholar
  33. 33.
    Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373PubMedCrossRefGoogle Scholar
  34. 34.
    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
  35. 35.
    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
  36. 36.
    Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813PubMedCrossRefGoogle Scholar
  37. 37.
    Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N (2010) Computer control of microscopes using microManager. Curr Protoc Mol Biol 14:14.20.1–14.20.17Google Scholar
  38. 38.
    McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133PubMedCrossRefGoogle Scholar
  39. 39.
    Sengupta P, Jovanovic-Talisman T, Skoko D, Renz M, Veatch SL, Lippincott-Schwartz J (2011) Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat Methods 8:969–975PubMedCrossRefGoogle Scholar
  40. 40.
    Murray JM, Appleton PL, Swedlow JR, Waters JC (2007) Evaluating performance in three-dimensional fluorescence microscopy. J Microsc 228:390–405PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Pakorn Kanchanawong
    • 1
  • Clare M. Waterman
    • 2
  1. 1.Department of Bioengineering, Mechanobiology InstituteNational University of SingaporeSingaporeSingapore
  2. 2.Cell Biology and Physiology CenterNational Heart Lung and Blood Institute, National Institutes of HealthBethesdaUSA

Personalised recommendations