Laurdan Generalized Polarization (GP) Spectroscopy

Membrane packing-sensitive fluorescence probe Laurdan (ex, 350 nm; em, 440/490 nm) when exposed to water molecules experiences solvent relaxation, resulting in a red shift.⁵⁵ The fluorescence intensities of Laurdan incorporated within lipid vesicles, with and without Rifabutin, were recorded using a temperature-controlled Varian Cary Eclipse fluorescence spectrophotometer, considering the range of temperature from 5 to 90 °C, giving a period of 3 min for equilibration with an accuracy of ±0.1 °C.

The use of the generic formula of (where I is the intensity at specified wavelengths) for LUVs containing complex lipid mixtures might not be a good indication to point out the changes imparted to the lipid head groups. Hence, GP was obtained from the spectra after log-normal spectral decomposition.^29,56 Each spectrum of Laurdan was treated as a superposition of two log-normal (LN) functions—one of each representing the two excited states of Laurdan: the nonrelaxed (blue channel) and the relaxed (green channel) states.

The raw data at each temperature was converted to fit in the wavenumber format—with wavenumber on the x-axis and intensity corresponding to the wavenumber (I = I_λ × λ², where I_λ is the intensity in wavelength scale and λ is the wavelength) on the y-axis. The spectra thus obtained are normalized, baseline-corrected, and then subjected log-normal deconvolution using Origin 2019b to two peaks (charge-transfer state, solvent relaxation state) or three peaks (also included locally excited state), and the two intensities corresponding to charge transfer (I_B for the blue channel) and solvent relaxation (I_G for the green channel) were picked. The GP curve is plotted using the following equation.