Improved Characterization of Raft-Mimicking Phase-Separation Phenomena in Lipid Bilayers Using Laurdan Fluorescence with Log-Normal Multipeak Analysis.

The study of biomimetic model membrane systems undergoing liquid-ordered (Lo)-liquid-disordered (Ld) phase separation using spectroscopic methods has played an important role in understanding the properties of lipid rafts in plasma membranes. In particular, the membrane-associated fluorescence probe Laurdan has proved to be a very efficient reporter of Lo-Ld phase separation in lipid bilayers using the general polarization (GP) parameter. A limitation of the GP approach is that it monitors only global average packing so that the contribution of each phase remains undetermined. The decomposition of Laurdan emission spectra has been proposed as an additional approach to overcoming this limitation. Here, further developments of this method for the study of Lo-Ld phase separation are described here for Laurdan in sphingomyelin-phosphatidylcholine-cholesterol large unilamellar vesicles. Lipid compositions corresponding to homogeneous Lo or Ld phases as well as undergoing thermally induced Lo-Ld phase separation were investigated. In addition, the occurrence of phase separation was checked by the fluorescence imaging of giant unilamellar vesicles. Decomposition into three log-normal components is used to show that an intermediate energy component is specifically associated with the occurrence of the Lo phase, with a small contribution from this component occurring above the phase-separation temperature being attributable to phase fluctuations. The ratio of the relative area of this intermediate-energy peak to that of the low-energy peak is shown to provide a straightforward index of Lo-Ld phase separation as a function of temperature, which is occasionally more sensitive than GP. It is also shown that can be used in conjunction with GP to gain further insight into Lo-Ld, the phase-separation processes. This latter feature is illustrated by the influence of the alcohol butanol on the Lo-Ld phase separation in sphingomyelin-phosphatidylcholine-cholesterol bilayers by showing that the effect of the alcohol occurs specifically at the onset of the phase separation, indicating a line tension mechanism. It is proposed that the three components of log-normal decomposition approaching Laurdan emission spectra provide a useful improvement for characterizing Lo-Ld phase-separation phenomena.