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Histochemistry and Cell Biology

, Volume 137, Issue 1, pp 1–10 | Cite as

Coordinate-based colocalization analysis of single-molecule localization microscopy data

  • Sebastian Malkusch
  • Ulrike Endesfelder
  • Justine Mondry
  • Márton Gelléri
  • Peter J. Verveer
  • Mike Heilemann
Original Paper

Abstract

Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.

Keywords

Colocalization Super-resolution microscopy Single-molecule fluorescence microscopy Cellular structures 

Notes

Acknowledgments

M.H. is grateful for funding by the German Ministry of Education and Research (BMBF; FORSYS initiative, grant nr. 0315262) and the German Science Foundation (DFG, grant nr. HE 6166/2-1). P.J.V is grateful for funding by the German Ministry of Education and Research (BMBF; FORSYS initiative, grant nr. 0315257) and by the German Science Foundation, as part of the NanoSci-ERA consortium (grant nr. VE 579/1-1).

References

  1. Adler J, Parmryd I (2010) Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander’s overlap coefficient. Cytom A 77:733–742CrossRefGoogle Scholar
  2. 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
  3. Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc Oxf 224:213–232CrossRefGoogle Scholar
  4. Churchman LS, Okten Z, Rock RS, Dawson JF, Spudich JA (2005) Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc Natl Acad Sci USA 102:1419–1423PubMedCrossRefGoogle Scholar
  5. Endesfelder U, Malkusch S, Flottmann B, Mondry J, Liguzinski P, Verveer PJ, Heilemann M (2011) Chemically induced photoswitching of fluorescent probes—a general concept for super-resolution microscopy. Molecules 16:3106–3118PubMedCrossRefGoogle Scholar
  6. Fehon RG, McClatchey AI, Bretscher A (2010) Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol 11:276–287PubMedCrossRefGoogle Scholar
  7. French AP, Mills S, Swarup R, Bennett MJ, Pridmore TP (2008) Colocalization of fluorescent markers in confocal microscope images of plant cells. Nat Protoc 3:619–628PubMedCrossRefGoogle Scholar
  8. Grecco HE, Verveer PJ (2011) FRET in cell biology: still shining in the age of super-resolution? Chemphyschem 12:484–490PubMedCrossRefGoogle Scholar
  9. Heilemann M (2010) Fluorescence microscopy beyond the diffraction limit. J Biotechnol 149:243–251PubMedCrossRefGoogle Scholar
  10. 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
  11. Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47:6172–6176PubMedCrossRefGoogle Scholar
  12. Koyama-Honda I, Ritchie K, Fujiwara T, Iino R, Murakoshi H, Kasai RS, Kusumi A (2005) Fluorescence imaging for monitoring the colocalization of two single molecules in living cells. Biophys J 88:2126–2136PubMedCrossRefGoogle Scholar
  13. Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF (2004a) A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 24:4070–4081PubMedCrossRefGoogle Scholar
  14. Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF (2004b) A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 24:4070–4081PubMedCrossRefGoogle Scholar
  15. McClatchey AI, Fehon RG (2009) Merlin and the ERM proteins—regulators of receptor distribution and signaling at the cell cortex. Trends Cell Biol 19:198–206PubMedCrossRefGoogle Scholar
  16. McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133Google Scholar
  17. Pertsinidis A, Zhang Y, Chu S (2010) Subnanometre single-molecule localization, registration and distance measurements. Nature 466:647–651PubMedCrossRefGoogle Scholar
  18. Radenovic A, Annibale P, Scarselli M, Kodiyan A (2010) Photoactivatable fluorescent protein mEos2 displays repeated photoactivation after a long-lived dark state in the red photoconverted form. J Phys Chem Lett 1:1506–1510CrossRefGoogle Scholar
  19. Ronneberger O, Baddeley D, Scheipl F, Verveer PJ, Burkhardt H, Cremer C, Fahrmeir L, Cremer T, Joffe B (2008) Spatial quantitative analysis of fluorescently labeled nuclear structures: problems, methods, pitfalls. Chromosome Res 16:523–562PubMedCrossRefGoogle Scholar
  20. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795PubMedCrossRefGoogle Scholar
  21. Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, Sedat JW (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332–1336PubMedCrossRefGoogle Scholar
  22. Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190:165–175PubMedCrossRefGoogle Scholar
  23. Smith CS, Joseph N, Rieger B, Lidke KA (2010) Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7:373–375PubMedCrossRefGoogle Scholar
  24. Wolter S, Endesfelder U, van de Linde S, Heilemann M, Sauer M (2011) Measuring localization performance of super-resolution algorithms on very active samples. Optics Express 19:7020–7033PubMedCrossRefGoogle Scholar
  25. Wombacher R, Heidbreder M, van de Linde S, Sheetz MP, Heilemann M, Cornish VW, Sauer M (2010) Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7:717–719PubMedCrossRefGoogle Scholar
  26. Zinchuk V, Zinchuk O (2008) Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr Protoc Cell Biol Chapter 4:4–19Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Sebastian Malkusch
    • 1
  • Ulrike Endesfelder
    • 1
  • Justine Mondry
    • 2
  • Márton Gelléri
    • 2
  • Peter J. Verveer
    • 2
  • Mike Heilemann
    • 1
  1. 1.Biotechnology and BiophysicsJulius-Maximilians-University WürzburgWürzburgGermany
  2. 2.Department of Systemic Cell BiologyMax Planck Institute of Molecular PhysiologyDortmundGermany

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