Abstract：
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 (So) to about 490 nm (green channel) in the liquid-crystalline phase (Lα). 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 So and Lα 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 So and in the blue channel in Lα exhibit a minimum near 90o. Surprisingly, the intensity in the green channel in Lα reaches a maximum near 90o. The fluorescence lifetimes in the two optical channels also reach a pronounced minimum near 90o in So and Lα, apart from the lifetime in the blue channel in Lα 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.
摘要：
溶剂化变色染料Laurdan广泛用于传感模型和生物膜的脂质堆积。荧光发射最大值从冷凝膜（So）中的约440nm（蓝色通道）移至液晶相（Lα）中的约490nm（绿色通道）。尽管基于荧光强度的广义偏振（GP）被广泛用于表征脂质膜，Laurdan的荧光寿命,在蓝色和绿色通道，较少用于此目的。在这里，我们通过对DMPC和DPPC的大型单层囊泡的So和Lα相的光谱测量来探索GP与荧光寿命之间的相关性。在两个脂质相的每个光学通道中观察到GP与寿命之间的正相关。在线性偏振的双光子激发下，在室温下对DPPC和DOPC的巨大单层囊泡进行微荧光测定，以使可能的Laurdan亚群以低于光学分辨率的比例分开。荧光强度,GP和荧光寿命取决于激发光的线性偏振的取向与光学横截面的膜的局部法线之间的角度。该角度变化取决于脂相和发射通道。So中的蓝色和绿色通道以及Lα中的蓝色通道中的GP和荧光强度在90o附近表现出最小值。令人惊讶的是,在Lα的绿色通道中的强度在90o附近达到最大值。在So和Lα中，两个光学通道中的荧光寿命也在90o附近达到明显的最小值，除了在Lα中的蓝色通道中的寿命之外，在Lα中，寿命很短，角度变化最小。据我们所知,这些实验观察首次证明了脂质膜中存在Laurdan的弯曲构象，正如先前分子动力学计算所建议的那样。
