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

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 moved laterally with kHz frequency through the membrane and the photon events 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-colour modality.