Department of Physics , Chalmers University of Technology , 412 96 Göteborg , Sweden.
Department of Chemistry and Biochemistry , Freie Universität Berlin , Berlin 14195 , Germany.
ACS Nano. 2018 Apr 24;12(4):3272-3279. doi: 10.1021/acsnano.7b07854. Epub 2018 Mar 19.
Fluorescence correlation spectroscopy (FCS) has provided a wealth of information on the composition, structure, and dynamics of cell membranes. However, it has proved challenging to reach the spatial resolution required to resolve biophysical interactions at the nanometer scale relevant to many crucial membrane processes. In this work, we form artificial cell membranes on dimeric, nanoplasmonic antennas, which shrink the FCS probe volume down to the ∼20 nm length scale. By analyzing the autocorrelation functions associated with the fluorescence bursts from individual fluorescently tagged lipids moving through the antenna "hotspots", we show that the confinement of the optical readout volume below the diffraction limit allows the temporal resolution of FCS to be increased by up to 3 orders of magnitude. Employing this high spatial and temporal resolution to probe diffusion dynamics of individual dye-conjugated lipids, we further show that lipid molecules diffuse either as single entities or as pairs in the presence of calcium ions. Removal of calcium ions by addition of the chelator EDTA almost completely removes the complex contribution, in agreement with previous theoretical predications on the role of calcium ions in mediating transient interactions between zwitterionic lipids. We envision that antenna-enhanced FCS with single-molecule burst analysis will enable resolving a broad range of challenging membrane biophysics questions, such as stimuli-induced lipid clustering and membrane protein dynamics.
荧光相关光谱学（FCS）为细胞膜的组成、结构和动力学提供了丰富的信息。然而，要达到解析与许多关键膜过程相关的纳米尺度生物物理相互作用所需的空间分辨率，一直具有挑战性。在这项工作中，我们在二聚体纳米等离子体天线上形成人工细胞膜，将 FCS 探针体积缩小到 ∼20nm 的长度尺度。通过分析单个荧光标记的脂质通过天线“热点”移动时产生的荧光爆发的自相关函数，我们表明光学读出体积的限制低于衍射极限，可以将 FCS 的时间分辨率提高 3 个数量级。利用这种高空间和时间分辨率来探测单个染料偶联脂质的扩散动力学，我们进一步表明，在钙离子存在的情况下，脂质分子要么以单个实体扩散，要么以对扩散。通过添加螯合剂 EDTA 去除钙离子几乎完全消除了复杂的贡献，这与钙离子在介导两性离子脂质之间瞬时相互作用中的作用的先前理论预测一致。我们设想，具有单分子爆发分析的天线增强 FCS 将能够解决一系列具有挑战性的膜生物物理问题，例如刺激诱导的脂质聚集和膜蛋白动力学。
