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

TL;DR

This paper introduces a method for scanning fluorescence correlation spectroscopy (SFCS) with a perpendicular scan path to measure membrane component diffusion, supported by open source software tools for data analysis.

Contribution

It presents a novel SFCS approach with minimal hardware needs and provides open source software for data processing and analysis.

Findings

Successful measurement of diffusion in biomembranes

Open source tools enable comprehensive data analysis

Method applicable in one- and two-focus, single- and dual-color modes

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.