Generalized polarization and time-resolved fluorescence provide evidence for different populations of Laurdan in lipid vesicles

• Published in: Journal of photochemistry and photobiology. B, Biology Vol. 250; p. 112833
• Main Authors: Bacalum, Mihaela, Radu, Mihai, Osella, Silvio, Knippenberg, Stefan, Ameloot, Marcel
• Format: Journal Article
• Language: English
• Published: Switzerland 01-01-2024
• Subjects: Fluorescence lifetimes; Laurdan; Angular photoselectivity; Generalized polarization; Conformational changes; Microheterogeneity
• ISSN: 1011-1344; 1873-2682
• DOI: 10.1016/j.jphotobiol.2023.112833

The solvatochromic dye Laurdan is widely used in sensing the lipid packing of both model and biological membranes. The fluorescence emission maximum shifts from about 440 nm (blue channel) in condensed membranes (S
) to about 490 nm (green channel) in the liquid-crystalline phase (L
). Although the fluorescence intensity based generalized polarization (GP) is widely used to characterize lipid membranes, the fluorescence lifetime of Laurdan, in the blue and the green channel, is less used for that purpose. Here we explore the correlation between GP and fluorescence lifetimes by spectroscopic measurements on the S
and L
phases of large unilamellar vesicles of DMPC and DPPC. A positive correlation between GP and the lifetimes is observed in each of the optical channels for the two lipid phases. Microfluorimetric determinations on giant unilamellar vesicles of DPPC and DOPC at room temperature are performed under linearly polarized two-photon excitation to disentangle possible subpopulations of Laurdan at a scale below the optical resolution. Fluorescence intensities, GP and fluorescence lifetimes depend on the angle between the orientation of the linear polarization of the excitation light and the local normal to the membrane of the optical cross-section. This angular variation depends on the lipid phase and the emission channel. GP and fluorescence intensities in the blue and green channel in S
and in the blue channel in L
exhibit a minimum near 90
. Surprisingly, the intensity in the green channel in L
reaches a maximum near 90
. The fluorescence lifetimes in the two optical channels also reach a pronounced minimum near 90
in S
and L
, apart from the lifetime in the blue channel in L
where the lifetime is short with minimal angular variation. To our knowledge, these experimental observations are the first to demonstrate the existence of a bent conformation of Laurdan in lipid membranes, as previously suggested by molecular dynamics calculations.