Lipid diffusion within black lipid membranes measured with dual-focus fluorescence correlation spectroscopy

We present an overview of the application of dual-focus fluorescence correlation spectroscopy (2f-FCS) for the measurement of diffusion coefficients within free-standing lipid membranes. The first part gives a detailed theoretical analysis of the expected performance of 2f-FCS, in particular about the sensitivity of the method with regard to precise focus position and to aberrations caused by refractive index mismatch or cover slide thickness deviation. After describing the experimental details of the 2f-FCS setup and the preparation of free-standing black lipid membranes (BLMs), we apply the method to study the diffusion of lipids within BLMs as a function of lipid composition and of ion valency and ionic strength of the surrounding buffer.