Laurdan Adopts Distinct, Phase-Specific Orientations in Lipid Membranes

For over 40 years, Laurdan has been widely used as a universal fluorescent probe for the study of lipid membranes. However, recent molecular dynamics simulations have uncovered previously unknown properties of Laurdan, revealing that it can adopt distinct conformations within the lipid bilayer, thereby influencing its molecular orientation. Despite these findings, experimental and quantitative validation has been lacking. Here, we present the first experimental study of the orientation of Laurdan in a phase-separated supported lipid bilayer, directly linking its spatial orientation to its emission spectra in liquid-ordered (L_o) and liquid-disordered (L_d) phases. Using azimuthally and radially polarized excitation beams, we show that in the L_o phase, Laurdan molecules align more parallel to the membrane normal, whereas in the L_d phase, they adopt a more planar orientation within the membrane. Interestingly, the emission spectra for both excitation modes converge at shorter wavelengths, but show deviations at longer wavelengths, particularly in the L_d phase. By refining our understanding of the behavior of Laurdan in lipid membranes, this study underlines the critical role of the molecular orientation of the dye in fluorescence-based membrane studies and highlights the need for orientation-sensitive analysis in biophysical investigations.