Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, USA.
Biophys J. 2013 Mar 19;104(6):1238-47. doi: 10.1016/j.bpj.2012.12.057.
Detection of the fluorescent properties of Laurdan has been proven to be an efficient tool to investigate membrane packing and ordered lipid phases in model membranes and living cells. Traditionally the spectral shift of Laurdan's emission from blue in the ordered lipid phase of the membrane (more rigid) toward green in the disordered lipid phase (more fluid) is quantified by the generalized polarization function. Here, we investigate the fluorescence lifetime of Laurdan at two different emission wavelengths and find that when the dipolar relaxation of Laurdan's emission is spectrally isolated, analysis of the fluorescence decay can distinguish changes in membrane fluidity from changes in cholesterol content. Using the phasor representation to analyze changes in Laurdan's fluorescence lifetime we obtain two different phasor trajectories for changes in polarity versus changes in cholesterol content. This gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content and thus perform a more comprehensive analysis of cell membrane heterogeneity. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in the membrane water content during cell migration.
已经证明，劳丹胺的荧光性质检测是一种研究模型膜和活细胞中膜包装和有序脂质相的有效工具。传统上，通过广义极化函数来量化劳丹胺在膜的有序脂质相（更刚性）中从蓝色到无序脂质相（更流动）的发射光谱位移。在这里，我们研究了劳丹胺在两个不同发射波长下的荧光寿命，发现当劳丹胺发射的偶极弛豫被光谱分离时，荧光衰减的分析可以区分膜流动性的变化和胆固醇含量的变化。使用相图表示法分析劳丹胺荧光寿命的变化，我们得到了极性变化和胆固醇含量变化的两个不同的相图轨迹。这使我们能够分辨具有不同特性的活细胞膜，如含水量和胆固醇含量，从而对细胞膜异质性进行更全面的分析。我们使用劳丹胺作为生物传感器来监测细胞迁移过程中膜含水量的变化，在 NIH3T3 细胞中演示了这种分析。
