Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.