Scanning Fluorescence Correlation Spectroscopy (SFCS) with a Scan Path Perpendicular to the Membrane Plane

  • Paul Müller
  • Petra Schwille
  • Thomas Weidemann
Part of the Methods in Molecular Biology book series (MIMB, volume 1076)

Abstract

Scanning fluorescence correlation spectroscopy (SFCS) with a scan path perpendicular to the membrane plane was introduced to measure diffusion and interactions of fluorescent components in free-standing biomembranes. Using a confocal laser scanning microscope (CLSM), the open detection volume is repeatedly scanned through the membrane at a kHz frequency. The fluorescence photons emitted from the detection volume are continuously recorded and stored in a file. While the accessory hardware requirements for a conventional CLSM are minimal, data evaluation can pose a bottleneck. The photon events must be assigned to each scan, in which the maximum signal intensities have to be detected, binned, and aligned between the scans, in order to derive the membrane-related intensity fluctuations of one spot. Finally, this time-dependent signal must be correlated and evaluated by well-known FCS model functions. Here we provide two platform-independent, open source software tools (PyScanFCS and PyCorrFit) that allow to perform all of these steps and to establish perpendicular SFCS in its one- or two-focus as well as its single- or dual-color modality.

Key words

Scanning fluorescence correlation spectroscopy (SFCS) Fluorescence correlation spectroscopy (FCS) Fluorescence cross-correlation spectroscopy (FCCS) Membrane diffusion Giant unilamellar vesicles (GUV) Diffusion Protein–protein interaction Ligand binding 

Notes

Acknowledgments

We thank Fabian Heinemann and Eugene Petrov for helpful discussions.

References

  1. 1.
    Kim SA, Heinze KG, Schwille P (2007) Fluorescence correlation spectroscopy in living cells. Nat Methods 4:963–973PubMedCrossRefGoogle Scholar
  2. 2.
    Liu P, Ahmed S, Wohland T (2008) The F-techniques: advances in receptor protein studies. Trends Endocrinol Metab 19:181–190PubMedCrossRefGoogle Scholar
  3. 3.
    Ries J, Schwille P (2006) Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy. Biophys J 91:1915–1924PubMedCrossRefGoogle Scholar
  4. 4.
    Ruan Q, Cheng MA, Levi M, Gratton E, Mantulin WW (2004) Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS). Biophys J 87:1260–1267PubMedCrossRefGoogle Scholar
  5. 5.
    Heinemann F, Betaneli V, Thomas FA, Schwille P (2012) Quantifying lipid diffusion by fluorescence correlation spectroscopy: a critical treatise. Langmuir 28(37):13395–133404PubMedCrossRefGoogle Scholar
  6. 6.
    Ries J, Chiantia S, Schwille P (2009) Accurate determination of membrane dynamics with line-scan FCS. Biophys J 96:1999–2008PubMedCrossRefGoogle Scholar
  7. 7.
    Ries J, Schwille P (2008) New concepts for fluorescence correlation spectroscopy on membranes. Phys Chem Chem Phys 10:3487–3497PubMedCrossRefGoogle Scholar
  8. 8.
    Dertinger T, Pacheco V, von der Hocht I, Hartmann R, Gregor I, Enderlein J (2007) Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. Chemphyschem 8:433–443PubMedCrossRefGoogle Scholar
  9. 9.
    Ries J, Yu S, Burkhardt M, Brand M, Schwille P (2009) Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms. Nat Methods 6:643–645PubMedCrossRefGoogle Scholar
  10. 10.
    Worch R, Bökel C, Höfinger S, Schwille P, Weidemann T (2010) Focus on composition and interaction potential of single-pass transmembrane domains. Proteomics 10:4196–4208PubMedCrossRefGoogle Scholar
  11. 11.
    Petrášek Z, Schwille P (2008) Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy. Biophys J 94:1437–1448PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Paul Müller
    • 1
  • Petra Schwille
    • 1
    • 2
  • Thomas Weidemann
    • 1
    • 2
  1. 1.BIOTEC, BiophysicsTechnische Universität DresdenDresdenGermany
  2. 2.Cellular and Molecular BiophysicsMax Planck Institute of BiochemistryMartinsriedGermany

Personalised recommendations