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Z-Scan Fluorescence Correlation Spectroscopy as a Tool for Diffusion Measurements in Planar Lipid Membranes

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Fluorescence Spectroscopy and Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1076))

Abstract

Studies of lateral diffusion are used for the characterization of the dynamics of biological membranes. One of the techniques that can be used for this purpose is fluorescence correlation spectroscopy (FCS), which belongs to the single-molecule techniques. Unfortunately, FCS measurements, when performed in planar lipid systems, are associated with a few sources of inaccuracy in the determination of the lateral diffusion coefficient. The main problems are related to the imperfect positioning of the laser focus relative to the plane of the sample. Another source of inaccuracy is the requirement for external calibration of the detection volume size. This protocol introduces a calibration-free method called Z-scan fluorescence correlation spectroscopy (Z-scan FCS), which is based on the determination of the diffusion time and particle number in steps along the optical (z-) axis by sequential FCS measurements. Z-scan FCS could be employed for diffusion measurements in planar membrane model systems—supported phospholipid bilayers (SPBs) and giant unilamellar vesicles (GUVs) and also in biological membranes. A result from measurements in SPBs is also presented in the protocol as a principle example of the Z-scan technique.

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References

  1. Elson E, Magde D (1974) Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13:1–27

    Article  CAS  Google Scholar 

  2. Magde D, Webb WW, Elson E (1972) Thermodynamic fluctuations in a reacting system–measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705–708

    Article  CAS  Google Scholar 

  3. Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61

    Article  PubMed  CAS  Google Scholar 

  4. Štefl M, Kułakowska A, Hof M (2009) Simultaneous characterization of lateral lipid and prothrombin diffusion coefficients by z-scan fluorescence correlation spectroscopy. Biophys J 97:L01–L03

    Article  PubMed  Google Scholar 

  5. Enderlein J, Gregor I, Patra D et al (2005) Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration. Chemphyschem 6:2324–2336

    Article  PubMed  CAS  Google Scholar 

  6. Enderlein J, Gregor I, Patra D et al (2004) Art and artefacts of fluorescence correlation spectroscopy. Curr Pharm Biotechnol 5:155–161

    Article  PubMed  CAS  Google Scholar 

  7. Thompson NL (1991) Fluorescence correlation spectroscopy. In: Lakowicz JR (ed) Topics in fluorescence spectroscopy. Vol 1 Plenum, New York, pp 337–378

    Google Scholar 

  8. Beneš M, Billy D, Hermens WT et al (2002) Muscovite (mica) allows the characterisation of supported bilayers by ellipsometry and confocal fluorescence correlation spectroscopy. Biol Chem 383:337–341

    PubMed  Google Scholar 

  9. Dertinger T, Pacheco V, von der Hocht I et al (2007) Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. Chemphyschem 8:433–443

    Article  PubMed  CAS  Google Scholar 

  10. Miszta A, Macháň R, Hermens WT et al (2010) Peptide-membrane interactions studied by ellipsometry, laser scanning microscopy, and z-scan fluorescence correlation spectroscopy. In: Castanho MA (ed) Membrane-active peptides: methods and results on structure and function. International University Line, California, pp 217–245

    Google Scholar 

  11. Gregor I, Patra D, Enderlein J (2005) Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation. Chemphyschem 6:164–170

    Article  PubMed  CAS  Google Scholar 

  12. Enderlein J, Gregor I, Patra D et al (2005) Statistical analysis of diffusion coefficient determination by fluorescence correlation spectroscopy. J Fluoresc 15:415–422

    Article  PubMed  CAS  Google Scholar 

  13. Benda A, Fagul’ová V, Deyneka A et al (2006) Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research. Langmuir 22:9580–9585

    Article  PubMed  CAS  Google Scholar 

  14. Enderlein J (1991) Autowave propagation in a piecewise linearized schlögl model with inhomogeneously distributed catalyst. Phys Lett A 156:429–435

    Article  CAS  Google Scholar 

  15. Enderlein J (2000) Theoretical study of detection of a dipole emitter through an objective with high numerical aperture. Opt Lett 25:634–636

    Article  PubMed  CAS  Google Scholar 

  16. Enderlein J, Ambrose WP (1997) Optical collection efficiency function in single-molecule detection experiments. Appl Opt 36:5298–5302

    Article  PubMed  CAS  Google Scholar 

  17. Benda A, Beneš M, Mareček V et al (2003) How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir 19:4120–4126

    Article  CAS  Google Scholar 

  18. Wohland T, Rigler R, Vogel H (2001) The standard deviation in fluorescence correlation spectroscopy. Biophys J 80:2987–2999

    Article  PubMed  CAS  Google Scholar 

  19. Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New York

    Book  Google Scholar 

  20. Ries J, Schwille P (2008) New concepts for fluorescence correlation spectroscopy on membranes. Phys Chem Chem Phys 10:3487–3497

    Article  PubMed  CAS  Google Scholar 

  21. Hess ST, Huang S, Heikal AA et al (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 41:697–705

    Article  PubMed  CAS  Google Scholar 

  22. Štefl M, Macháň R, Hof M (2011) Z-scan fluorescence correlation spectroscopy: a powerful tool for determination of lateral diffusion in biological systems. In: Geddes CD (ed) Reviews in fluorescence 2009. Reviews in fluorescence. Springer, New York, pp 321–344

    Google Scholar 

  23. Schwille P, Oehlenschlager F, Walter N (1996) Quantitative hybridization kinetics of dna probes to rna in solution followed by diffusional fluorescence correlation analysis. Biochemistry 35:10182–10193

    Article  PubMed  CAS  Google Scholar 

  24. Widengren J, Mets U, Rigler R (1995) Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J Phys Chem 99:13368–13379

    Article  CAS  Google Scholar 

  25. Macháň R, Hof M (2010) Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy. BBA-Biomembranes 1798:1377–1391

    Article  PubMed  Google Scholar 

  26. Donsmark J, Rischel C (2007) Fluorescence correlation spectroscopy at the oil-water interface: hard disk diffusion behavior in dilute beta-lactoglobulin layers precedes monolayer formation. Langmuir 23:6614–6623

    Article  PubMed  CAS  Google Scholar 

  27. Provencher SW (1982) A constrained regularization method for inverting data represented by linear algebraic or integral equations. Comput Phys Commun 27:213–227

    Article  Google Scholar 

  28. Enderlein J (2005) Dependence of the optical saturation of fluorescence on rotational diffusion. Chem Phys Lett 410:452–456

    Article  CAS  Google Scholar 

  29. Przybylo M, Sýkora J, Humpolíčková J et al (2006) Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions. Langmuir 22:9096–9099

    Article  PubMed  CAS  Google Scholar 

  30. Ries J, Schwille P (2006) Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy. Biophys J 91:1915–1924

    Article  PubMed  CAS  Google Scholar 

  31. Chiantia S, Ries J, Kahya N et al (2006) Combined afm and two-focus sfcs study of raft-exhibiting model membranes. Chemphyschem 7:2409–2418

    Article  PubMed  CAS  Google Scholar 

  32. Humpolíčková J, Gielen E, Benda A et al (2006) Probing diffusion laws within cellular membranes by z-scan fluorescence correlation spectroscopy. Biophys J 91:L23–L25

    Article  PubMed  Google Scholar 

  33. Milon S, Hovius R, Vogel H et al (2003) Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments. Chem Phys 288:171–186

    Article  CAS  Google Scholar 

  34. Palmer AG, Thompson NL (1989) Optical spatial intensity profiles for high order autocorrelation in fluorescence spectroscopy. Appl Opt 28:1214–1220

    Article  CAS  Google Scholar 

  35. Sorscher SM, Klein MP (1980) Profile of a focused collimated laser beam near the focal minimum characterized by fluorescence correlation spectroscopy. Rev Sci Instrum 51:98–102

    Article  CAS  Google Scholar 

  36. Macháň R, Hof M (2010) Recent developments in fluorescence correlation spectroscopy for diffusion measurements in planar lipid membranes. Int J Mol Sci 11:427–457

    Article  PubMed  Google Scholar 

  37. Wawrezinieck L, Rigneault H, Marguet D et al (2005) Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Biophys J 89:4029–4042

    Article  PubMed  CAS  Google Scholar 

  38. Monincová L, Buděšínský M, Slaninová J et al (2010) Novel antimicrobial peptides from the venom of the eusocial bee halictus sexcinctus (hymenoptera: halictidae) and their analogs. Amino Acids 39:763–775

    Article  PubMed  Google Scholar 

  39. Macháň R (2012) Supported lipid bilayers and antimicrobial peptides: characterized by ellipsometry and fluorescence correlation spectroscopy. LAP LAMBERT Academic, Germany

    Google Scholar 

  40. Guo L, Har JY, Sankaran J et al (2008) Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study. Chemphyschem 9:721–728

    Article  PubMed  CAS  Google Scholar 

  41. Beneš M, Billy D, Benda A et al (2004) Surface-dependent transitions during self-assembly of phospholipid membranes on mica, silica, and glass. Langmuir 20:10129–10137

    Article  PubMed  Google Scholar 

  42. Kask P, Gunther R, Axhausen P (1997) Statistical accuracy in fluorescence fluctuation experiments. Eur Biophys J Biophys Lett 25:163–169

    Article  Google Scholar 

  43. Forstner MB, Yee CK, Parikh AN et al (2006) Lipid lateral mobility and membrane phase structure modulation by protein binding. J Am Chem Soc 128:15221–15227

    Article  PubMed  CAS  Google Scholar 

  44. Rüttinger S, Buschmann V, Krämer B et al (2008) Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy. J Microsc 232:343–352

    Article  PubMed  Google Scholar 

  45. Böhmer M, Wahl M, Rahn HJ et al (2002) Time-resolved fluorescence correlation spectroscopy. Chem Phys Lett 353:439–445

    Article  Google Scholar 

  46. Kapusta P, Wahl M, Benda A et al (2007) Fluorescence lifetime correlation spectroscopy. J Fluoresc 17:43–48

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The authors thank Martin Štefl for his comments and suggestions and collaborators from Institute of Organic Chemistry and Biochemistry of ASCR for providing halictine peptide. This work was supported by Czech Science Foundation through Grant No. P208/10/0376 and by Ministry of Education, Youth and Sports of the Czech Republic via BIO-OPT-XUV Research Team Advancement at the Faculty of Biomedical Engineering, Czech Technical University in Prague, registration nr.: MEYS ESF Project CZ.1.07/2.3.00/20.0092. M.H. acknowledges financial support by AS CR via Praemium Academiae award.

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Authors and Affiliations

  1. Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic

    Tomáš Steinberger & Radek Macháň

  2. J. Heyrovský Institute of Physical Chemistry v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Tomáš Steinberger, Radek Macháň & Martin Hof

Authors
  1. Tomáš Steinberger
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  2. Radek Macháň
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  3. Martin Hof
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Editors and Affiliations

  1. KULeuven-Biomolecular Dynamics BioSCENTer, Leuven, Belgium

    Yves Engelborghs

  2. MicroSpectroscopy Centre, University of Wageningen Biochemistry, Wageningen, The Netherlands

    Antonie J.W.G. Visser

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Steinberger, T., Macháň, R., Hof, M. (2014). Z-Scan Fluorescence Correlation Spectroscopy as a Tool for Diffusion Measurements in Planar Lipid Membranes. In: Engelborghs, Y., Visser, A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology, vol 1076. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-649-8_28

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  • DOI: https://doi.org/10.1007/978-1-62703-649-8_28

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-648-1

  • Online ISBN: 978-1-62703-649-8

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