Benda Ales, Fagul'ová Veronika, Deyneka Alexander, Enderlein Joerg, Hof Martin
J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejskova 3, 18223 Praha 8, Czech Republic.
Langmuir. 2006 Nov 7;22(23):9580-5. doi: 10.1021/la061573d.
A new concept based on fluorescence lifetime correlation spectroscopy (FLCS) is presented allowing the simultaneous determination of diffusion coefficients of identical molecules located in different environments. The difference in fluorescence lifetimes, which is the main prerequisite for FLCS, is reached by locating one population of the dye close to a light-absorbing surface. Since such surfaces quench fluorescence, the fluorescence lifetime of chromophores located close to these surfaces can be tuned in a specific manner. This approach has been demonstrated for a BODIPY-tail-labeled lipid in supported phospholipid bilayers (SPBs) as well as in phospholipid multilayers adsorbed onto solid supports. In particular, the effect of the solid support type on the fluorescence lifetime as well as its dependence on the BODIPY-support distance has been characterized and verified by theoretical considerations based on precise determination of refractive indices of the used supports. While the fluorescence lifetime of BODIPY dye is 5.6 ns in small unilamellar vesicles (SUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), the lifetime is 1.8 ns in DOPC/DOPS SPBs adsorbed onto ITO-covered glass or 3.0 ns in a DOPC/DOPS monolayer adsorbed onto seven 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) layers on oxidized silicon. Using these particular systems, we demonstrated that FLCS enables one to characterize simultaneously two-dimensional lipid diffusion in the planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers using single dye labeling. The autocorrelation functions obtained by this new approach do agree with those obtained by standard FCS on isolated SPBs or vesicles. Possible applications of this virtual two-channel measurement using single dye labeling as well as one detection channel are discussed.
提出了一种基于荧光寿命相关光谱（FLCS）的新概念，可同时测定位于不同环境中的相同分子的扩散系数。通过将一部分染料定位在靠近光吸收表面的位置，可实现FLCS的主要前提条件——荧光寿命的差异。由于此类表面会淬灭荧光，靠近这些表面的发色团的荧光寿命可通过特定方式进行调节。该方法已在支持的磷脂双层（SPB）以及吸附在固体支持物上的磷脂多层膜中的BODIPY尾标记脂质上得到了验证。特别是，基于对所用支持物折射率的精确测定，通过理论考量对固体支持物类型对荧光寿命的影响及其对BODIPY-支持物距离的依赖性进行了表征和验证。在由1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）和1,2-二油酰基-sn-甘油-3-[磷酸-L-丝氨酸]（DOPS）组成的小单层囊泡（SUV）中，BODIPY染料的荧光寿命为5.6 ns，而在吸附在ITO覆盖玻璃上的DOPC/DOPS SPB中，寿命为1.8 ns，或在吸附在氧化硅上的七个1,2-二棕榈酰基-sn-甘油-3-磷酸（DPPA）层上的DOPC/DOPS单层中，寿命为3.0 ns。使用这些特定系统，我们证明了FLCS能够使用单染料标记同时表征平面脂质层中的二维脂质扩散和脂质层上方本体中的三维囊泡扩散。通过这种新方法获得的自相关函数与通过标准FCS在分离的SPB或囊泡上获得的自相关函数一致。讨论了使用单染料标记以及一个检测通道进行这种虚拟双通道测量的可能应用。
