Gielen Ellen, Smisdom Nick, vandeVen Martin, De Clercq Ben, Gratton Enrico, Digman Michelle, Rigo Jean-Michel, Hofkens Johan, Engelborghs Yves, Ameloot Marcel
Laboratory for Cell Physiology, Biomedical Research Institute, Hasselt University and transnationale Universiteit Limburg, Agoralaan, Bldg C, B-3590 Diepenbeek, Belgium.
Langmuir. 2009 May 5;25(9):5209-18. doi: 10.1021/la8040538.
The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope (CLSM), and the wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The original introduction of RICS used two-photon excitation and photon counting detection. However, most CLSM systems are based on one-photon excitation with analog detection. Here we report on the performance of such a commercial CLSM (Zeiss LSM 510 META) in the study of the diffusion of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indodicarbocyanine perchlorate (DiI-C(18)(5)) both in giant unilamellar vesicles and in the plasma membrane of living oligodendrocytes, i.e., the myelin-producing cells of the central nervous system. It is shown that RICS on a commercial CLSM with analog detection allows for reliable results in the study of membrane diffusion by removal of unwanted correlations introduced by the analog detection system. The results obtained compare well with those collected by FRAP and FCS.
细胞膜在组成和相互作用方面的异质性导致其成分具有广泛的扩散系数。因此，必须应用几种互补的显微荧光技术，如荧光相关光谱法（FCS）、光漂白后荧光恢复法（FRAP）和单粒子追踪法（SPT）来探索膜成分的动力学。最近引入的光栅图像相关光谱法（RICS）比这些方法单独使用时具有更宽的动态范围，并且能够对动态特性进行空间映射。RICS是在共聚焦激光扫描显微镜（CLSM）上实现的，通过利用扫描激光束在生成共聚焦图像时携带的固有时间信息来实现宽动态范围。RICS最初的引入使用了双光子激发和光子计数检测。然而，大多数CLSM系统基于单光子激发和模拟检测。在这里，我们报告了这样一台商用CLSM（蔡司LSM 510 META）在研究荧光脂质类似物1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（DiI-C(18)(5)）在巨大单层囊泡和活少突胶质细胞（即中枢神经系统的髓磷脂生成细胞）质膜中的扩散时的性能。结果表明，具有模拟检测功能的商用CLSM上的RICS通过消除模拟检测系统引入的不必要相关性，能够在膜扩散研究中获得可靠结果。所得结果与通过FRAP和FCS收集的结果相当。
