Factors influencing fluorescence correlation spectroscopy measurements on membranes: Simulations and experiments

Abstract

Fluorescence correlation spectroscopy (FCS) is a widely used technique for the measurement of diffusion coefficients and concentrations. The standard deviation (SD) of FCS in solution is normally smaller than 1-5%, but it is often 50100% or larger on cell membranes. This effect is usually attributed to the non-homogeneous structure of biological membranes that leads to variations of the diffusion coefficient at different membrane sites. Here we perform experiments and simulations to investigate the influence of geometrical configurations on the recovered diffusion coefficient. We find that this parameter can vary by factors of at least 2-6 depending on alignment of sample and focus, and on membrane topography. Misalignment and membrane topography increase the SD of membrane measurements and make it impossible to decide whether free diffusion or anomalous diffusion is measured. We propose a routine to optimally perform FCS measurements on membranes and to identify the source of the deviations. © 2003 Elsevier Science B.V. All rights reserved.