A new optical probe, di-4-ANEPPDHQ, for cholesterol enriched domains in lipid bilayer membranes

Here I present an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. Its emission spectrum blue shifts with increasing cholesterol concentration and in liquid-ordered phases relative to liquid-disordered phases. Given these optical properties, I use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and non-linear microscopies. The dye shows green and red fluorescence in liquid-ordered and disordered domains, respectively. I propose that this reflects the membrane rigidity difference in the two phases. I also observe stronger second harmonic generation signals in the liquid-disordered domains, consistent with higher affinity of the dye to the disordered phase domains. I further test the efficacy of the dye to report lipid domains in cell membranes in studies of polarized migrating neutrophils, sperms and fibroblast cells.