Time-resolved Laurdan fluorescence reveals insights into membrane viscosity and hydration levels

Membrane viscosity and hydration levels characterize the biophysical properties of biological membranes and are reflected in the rate and extent of solvent relaxation, respectively, of environmentally sensitive fluorophores such as Laurdan. Here we first developed a method for a time-resolved General Polarization (GP) analysis with fluorescence-lifetime imaging microscopy (FLIM) that captures both the extent and rate of Laurdan solvent relaxation. We then conducted time-resolved GP measurements with Laurdan-stained model membranes and cell membranes. These measurements revealed that cholesterol levels in lipid vesicles altered membrane hydration and viscosity while curvature had little effect on either parameter. We also applied the method to the plasma membrane of live cells using a critical angle fluorescence (SAF) objective, to our knowledge the first time FLIM images were generated with SAF. Here, we found that local variations in membrane cholesterol most likely account for the heterogeneity of Laurdan lifetime in plasma membrane. In conclusion, time-resolved GP measurements provide additional insights into the biophysical properties of membranes.