Biophysics Institute, Biotec/Technische Universität Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
Langmuir. 2012 Sep 18;28(37):13395-404. doi: 10.1021/la302596h. Epub 2012 Aug 31.
Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm(2) s(-1) at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.
荧光相关光谱（FCS）测量广泛用于确定生物膜中脂质和蛋白质的扩散系数。近年来，已经引入了几种 FCS 的变体。然而，迄今为止，还缺乏对这些方法在相同系统上的综合比较。此外，对于众所周知或广泛使用的膜系统，已经确定的扩散系数没有一致的值。本研究旨在通过确定由二油酰基磷脂酰胆碱（DOPC）制成的巨大单层囊泡（GUV）中荧光脂质类似物 1,1'-二辛基-3,3,3',3'-四甲基吲哚碳菁（DiD）的绝对扩散系数，为膜上的 FCS 实验的更好可比性做出贡献，该扩散系数可在未来的研究中用作参考值。为此，比较了采用不同校准方法的五种 FCS 变体。讨论了每种特定 FCS 方法的潜在误差源及其避免方法。对于所有研究的 FCS 变体，均获得了 DiD 在 DOPC 中的绝对扩散系数的良好一致性。在 23.5°C ± 1.5°C 时，获得了 D = 10.0 ± 0.4 μm(2) s(-1)的平均扩散系数。使用不同方法进行的独立验证表明，可以安全地将此值用于校准目的。此外，通过测量由 DOPC 和胆固醇组成的 GUV 中 DiD 的扩散系数，还验证了这些方法在缓慢扩散的情况下的可比性。
