Amaro Mariana, Šachl Radek, Jurkiewicz Piotr, Coutinho Ana, Prieto Manuel, Hof Martin
Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry of the Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic.
Centre for Molecular Chemistry and Physics and Instituto de Nanociência e Nanotecnologia, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Departamento Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal.
Biophys J. 2014 Dec 16;107(12):2751-2760. doi: 10.1016/j.bpj.2014.10.058.
Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.
荧光方法是获取生物膜动态和拓扑信息的通用工具，因为脂质膜中发生的分子相互作用通常与荧光发射发生在同一时间尺度上。特别是荧光强度衰减，是荧光团分子环境的有力报告者。荧光寿命可能对荧光团附近几纳米范围内的局部极性、水合作用、粘度和/或荧光猝灭剂/能量受体的存在敏感。本综述将给出一些示例，说明时间分辨荧光测量如何比时间积分（稳态）方法提供有关系统更有价值和详细的信息：1）使用时间相关光谱位移测定膜极性和流动性；2）通过荧光寿命成像显微镜识别亚微观结构域；3）通过染料自猝灭测定阐明膜泄漏机制；4）通过时间分辨Förster共振能量转移测量评估纳米结构域大小。
