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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.