Complementary Fluorescence Emission and Second Harmonic Spectra Improve Bilayer Characterization

Complementary investigation of Laurdan fluorescence emission and second harmonic (SH) spectra in nonpolar, protic and aprotic polar solvents and phospholipid bilayers was carried out. SH of spectra computed using methods familiar in electro spin resonance spectroscopy yielded better resolution. Spectra were fit to log-normal distributions. SH spectra showed presence of two emissions in protic polar and nonpolar solvents and in both bilayer gel and liquid phases and a single line in aprotic polar solvents. Each of the half maximal positions of each line in both homogenous solvents and bilayers, expresses similar linearity with peak position. This shared feature suggests planar and nonplanar Laurdan conformation respectively in the longer (red) and shorter (blue) wavelength emitting states. The weaker 432 nm blue line, not detected before in the gel phase, is distinguishable in the SH. Temperature trajectories of areas and peak positions of the individual lines bring new insight into the nature of lipid packing and evolution of domains, indicating inhomogeneous lipid packing even in the gel phase. The blue line identifies as emission from Laurdan in tighter packed regions and the dominant 445-448 nm red line in the gel phase shifting to 484 nm in the liquid phase as emission from Laurdan-water coupled states that are in varying stages of relaxation according to temperature and phase. Unexpected increase in the area of the blue line with temperature through the gel-liquid transition is consistent with coexisting low and high curvature domains and Laurdan's preference for less polar low curvature domains.