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)


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 



We thank Fabian Heinemann and Eugene Petrov for helpful discussions.


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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

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