Börner Richard, Ehrlich Nicky, Hohlbein Johannes, Hübner Christian G
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
Bionanotechnology and Nanomedicine Laboratory, University of Copenhagen, Universitetsparken 5, 2100, Copenhagen, Denmark.
J Fluoresc. 2016 May;26(3):963-75. doi: 10.1007/s10895-016-1784-5. Epub 2016 Mar 14.
Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, such that the use of fluorescent lipid dyes (DiO) allows to establish a a wide range of dipole orientations experimentally. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach.
单分子之间的相互作用在很大程度上取决于它们相互的三维取向。最近，我们展示了一种技术，该技术能够利用荧光在多个检测通道中的偏振分辨分布来确定单个偶极发射体的取向。由于该方法基于单光子检测，它还允许进行荧光相关光谱（FCS）以及动态各向异性测量，从而能够获取直至纳秒时间尺度的快速取向动力学信息。在诸如脂质膜等非各向同性环境中，三维取向尤其令人关注，而脂质膜在生物学中具有重要意义。我们使用标记有荧光染料直至单分子浓度的巨型单层囊泡（GUV）作为模型系统，来评估不同时间尺度下取向确定的稳健性并量化相关误差。这些囊泡提供了一个明确界定的球形表面，使得使用荧光脂质染料（DiO）能够在实验中建立广泛的偶极取向范围。为补充我们的实验数据，我们对掺入脂质膜中的偶极的旋转动力学进行了蒙特卡罗模拟。我们的研究以高时空分辨率提供了关于脂质膜中染料取向行为的全面视图，代表了一种六维荧光检测方法。
