Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

Abstract

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 microm(-2)) and very low surface concentrations (single-molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well-suited for the intermediate concentration range of about 0.1-100 microm(-2). However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z-scan FCS, is calibration-free for membrane measurements, but requires several experiments at different well-controlled focusing positions. A recently established FCS method, electron-multiplying charge-coupled-device-based total internal reflection FCS (TIR-FCS), referred to here as imaging TIR-FCS (ITIR-FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR-FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.

Fluorescence Techniques to Study Lipid Dynamics

“…There have been many studies on phase separated model membranes, supported or free-standing, to determine the diffusion characteristics of lipids in different phases (Chiantia et al 2008(Chiantia et al , 2009Lingwood et al 2008;. It has been shown that the diffusion coefficient is influenced by environmental conditions such as ionic strength or sugar content of the medium (Bockmann et al 2003;Sum et al 2003;Doeven et al 2005;van den Bogaart et al 2007;Guo et al 2008;Vacha et al 2009). The role of cholesterol in membrane organization, a big issue in lipid biology, has been intensively addressed by FCS Bacia et al 2004Bacia et al , 2005Kahya and Schwile 2006b).…”

Formation and fluidity measurement of supported lipid bilayer on polyvinyl chloride membrane

“…5. We fitted the recovery curves to eq (1) [30][31][32] and the mobile fraction of 97% showed formation of continuous single SLB. On the PVC membrane, the mobile fraction of 68% means that 32% of the SLB in the coverage were formed as isolated patches, or trapped by the substrate.…”

“…SiO 2 /Si, glass, mica, TiO 2 ) have smaller diffusion coefficients than free-standing lipid bilayers such as giant liposomes. [30][31][32][33] One possible explanation for the rapid diffusion in spite of the large immobile fraction (many defects) is the microscopic heterogeneity in the SLB on the PVC membrane: the lipid diffusion in the mobile region is as fast as that in freestanding membrane, though the lipids in the immobile regions are trapped on the surface or divided from the fluid and continuous SLB because of the strongly interaction with the PVC surface.…”

Model Bio‐Membranes Investigated by AFM and AFS: A Suitable Tool to Unravel Lipid Organization and their Interaction with Proteins

“…Concerning the comparison of the diff usion properties of SLBs with other lipid bilayer model systems, experimental results have shown that the lipid diff usion in free standing bilayers (GUV, Giant Unilamellar Vesicles) is more than two times faster than in supported lipid bilayers (the diff usion coeffi cient is D = 7.8 μm 2 s -1 for GUVs and D = 3.1 μm 2 s -1 for SLB on mica) [56][57][58]. Th e diff erence in the diff usion constant is usually attributed to the interaction of the substrate with the bilayer or to surface sticking of the lipids creating pinning points between the support and the bilayer.…”