4.5. Laurdan Generalized Polarization

Laurdan is an environment-sensitive fluorophore widely used to quantify the lipid packing. The polycyclic aromatic compound naphthalene possesses a dipole moment that causes reorientation of water molecules which results in a red-shift of the probe emission [59]. Accordingly, after excitation at 350 nm, the emission spectrum of Laurdan in phospholipid membranes presents two local maxima, one at about 440 nm for ordered lipid (gel) phases and the other around 490 nm for the more disordered lipid phases (liquid-crystalline). 0.2 mol% of Laurdan was added to the lipids dissolved in chloroform/methanol. The lipid film was resuspended in HEPES 5 mM at pH 7.5 with a final concentration of 1 mM. To precisely quantify the polarity change, we used the general polarization (GP) term [44,45]:

where I₄₄₀ and I₄₉₀ are the emission intensities at 440 and 490 nm, respectively. GP values can range from +1 to −1, i.e., −1 being the highest lipid membrane fluidity.

The temperature-dependent measurements were performed on a K2 multifrequency phase and modulation fluorimeter (ISS Inc. (Champaign, IL, USA)). We used a quartz cuvette with a volume of 100 μL in a temperature range of 5–90 °C. Temperature control was achieved using a circulating water bath with an accuracy of ±0.1 °C.