Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16/18, D-04107 Leipzig, Germany.
Methods Appl Fluoresc. 2022 Aug 24;10(4). doi: 10.1088/2050-6120/ac87ea.
Small lipid vesicles (with diameter ≤100 nm) with their highly curved membranes comprise a special class of biological lipid bilayers. The mechanical properties of such membranes are critical for their function, e.g. exocytosis. Cholesterol is a near-universal regulator of membrane properties in animal cells. Yet measurements of the effect of cholesterol on the mechanical properties of membranes have remained challenging, and the interpretation of such measurements has remained a matter of debate. Here we show that nanosecond fluorescence correlation spectroscopy (FCS) can directly measure the ns-microsecond rotational correlation time () of a lipid probe in high curvature vesicles with extraordinary sensitivity. Using a home-built 4-Pi fluorescence cross-correlation spectrometer containing polarization-modulating elements, we measure the rotational correlation time () of Nile Red in neurotransmitter vesicle mimics. As the cholesterol mole fraction increases from 0 to 50%,increases from 17 ± 1 to 112 ± 12 ns, indicating a viscosity change of nearly a factor of 7. These measurements are corroborated by solid-state NMR results, which show that the order parameter of the lipid acyl chains increases by about 50% for the same change in cholesterol concentration. Additionally, we measured the spectral parameters of polarity-sensitive fluorescence dyes, which provide an indirect measure of viscosity. The green/red ratio of Nile Red and the generalized polarization of Laurdan show consistent increases of 1.3× and 2.6×, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and a wider dynamic range compared to other conventional techniques. Significantly, we observe that the viscosity of neurotransmitter vesicle mimics is remarkably sensitive to their cholesterol content.
小脂质囊泡（直径≤100nm）具有高度弯曲的膜，它们构成了一类特殊的生物脂质双层。这些膜的力学性质对于它们的功能至关重要，例如胞吐作用。胆固醇是动物细胞中膜性质的一种近乎普遍的调节剂。然而，胆固醇对膜力学性质的影响的测量仍然具有挑战性，并且对这些测量的解释仍然存在争议。在这里，我们表明纳秒荧光相关光谱（FCS）可以直接测量具有高曲率的囊泡中脂质探针的纳秒-微秒旋转相关时间（τ），具有非凡的灵敏度。我们使用含有偏振调制元件的自制 4-Pi 荧光互相关光谱仪，测量神经递质囊泡模拟物中尼罗红的旋转相关时间（τ）。随着胆固醇摩尔分数从 0 增加到 50%，τ从 17±1 增加到 112±12ns，表明粘度变化近 7 倍。这些测量结果得到固态 NMR 结果的证实，固态 NMR 结果表明，对于胆固醇浓度相同的变化，脂质酰链的有序参数增加了约 50%。此外，我们测量了对极性敏感的荧光染料的光谱参数，这提供了粘度的间接测量。尼罗红的绿/红比和 Laurdan 的广义极化分别显示出 1.3×和 2.6×的一致增加。我们的结果表明，与其他传统技术相比，旋转 FCS 可以直接测量高度弯曲的膜的粘度，具有更高的灵敏度和更宽的动态范围。重要的是，我们观察到神经递质囊泡模拟物的粘度对其胆固醇含量非常敏感。
