Machán Radek, Hof Martin
J. Heyrovský Institute of Physical Chemistry v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Biochim Biophys Acta. 2010 Jul;1798(7):1377-91. doi: 10.1016/j.bbamem.2010.02.014. Epub 2010 Feb 24.
Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.
对生物膜及其人工模型中脂质横向流动性的研究提供了有关膜动力学和结构的信息；基于光学显微镜的方法对于此类研究非常方便。我们重点介绍荧光相关光谱法（FCS），解释其原理，并综述其诸如双焦点、Z扫描或扫描FCS等最新版本，这些版本克服了标准FCS的大多数假象（特别是那些因需要外部校准而产生的假象），使其成为一种可靠且通用的方法。还将FCS与单粒子追踪和荧光光漂白恢复进行了比较，并简要综述了这些方法的适用性和局限性。我们讨论了平面脂质膜中横向流动性研究的几个关键问题，即膜和水相组成（离子强度和糖含量）、荧光示踪分子的选择、两个膜小叶之间以及膜与固体支持物之间（在支持膜的情况下）的摩擦耦合或膜不均匀性的存在对脂质横向流动性的影响。综述了近期针对这些问题的FCS研究，并提及了对最终差异的可能解释。
