2.2.4. Fluorescence Spectroscopy of Laurdan-, DPH- and Di-8-ANEPPS-Labeled LUVs

The lipid structural order parameter of the bilayer in the fatty acid core and at the glycerol level are probed by fluorescence spectroscopy of DPH [29] and Laurdan [30], respectively. The excitation wavelength for Laurdan is 355 nm. In disordered lipid surroundings, its emission maximum with intensity *I*<sup>490</sup> is centered at 490 nm. In more ordered lipid environment, the maximum of Laurdan emission is shifted at 440 nm with intensity *I*440. The lipid packing is quantified by the parameter *GP* representing Laurdan generalized polarization accordingly [30]:

$$GP = \frac{I\_{440} - I\_{490}}{I\_{440} + I\_{490}} \tag{1}$$

It theoretically assumes values from −1, corresponding to disordered membrane up to 1 for most ordered molecules. We record three times all emission spectra from 390 to 600 nm, then average, and subtract background. For every studied LUV suspension, the emission spectrum of Laurdan is obtained and *GP* values are calculated in the temperature range (20 − 60) °C.

We apply 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence spectroscopy [31] to assess membrane fluidity as a function of sugar concentration in LUV samples. DPH represents a hydrocarbonic probe, which is nearly non-fluorescent in water. Contrastingly, in lipid membranes, it exhibits strong fluorescence. In our experiment, the excitation is adjusted to 358 nm. We record the emission at 430 nm. The fluorescence polarization is measured as described in [32]. The DPH fluorescence anisotropy *rDPH*, quantifying membrane fluidity is expressed by the intensities *I* of the polarized components of the fluorophore emission:

$$r\_{DPH} = \frac{I\_{VV} - GI\_{VH}}{I\_{VV} + 2GI\_{VH}} \,\text{,}\tag{2}$$

where *V* and *H* stand for "vertical" and "horizontal", corresponding to the orientation of the polarization axis with respect to the light direction. The first index indicates the polarization of the excitation light and the second one denotes the polarization of the emission signal, corresponding to the orientation of the excitation and emission polarizers, respectively. The grating factor *G* = *IHV*/*IHH* reflects the sensitivity of the instrument toward vertically and horizontally polarized light. Around the main phase transition of the bilayer, the fluorescence anisotropy of DPH sharply increases. It can assume values from −0.2 to 0.4 [31].

The membrane dipole potential in sugar-containing aqueous solutions is quantified by the fluorescence excitation ratio of the potential-sensitive fluorescent styryl dye di-8- ANEPPS. In the case of di-8-ANEPPS incorporated into LUVs, fluorescence is excited at 420 nm and 520 nm and detected at 670 nm. It has been shown that the fluorescence intensity ratio Rex = I<sup>670</sup> (exc.420)/I<sup>670</sup> (exc.520) is proportional to the dipole potential Ψ*<sup>d</sup>* , independently of fluidity effects [33–35]:

$$\mathbf{Y}\_d = (\mathbf{R}\_{\text{ex}} + 0.3) / 0.0043 \tag{3}$$

The dipole potential occurs transversally between the water–lipid interface and the hydrocarbon interior from the contribution of all polarized and polarizable chemical groups of lipid molecules and by the hydration shell of the bilayer [36].

FP-8300 spectrofluorimeter (Jasco) equipped with polarizers and a thermostatted (±0.1 °C) cuvette holder with quartz cuvettes is used. Samples are equilibrated for 5 min at the desired temperature. Excitation and emission slits are adjusted to 5 nm.
