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首页> 外文期刊>Langmuir: The ACS Journal of Surfaces and Colloids >Measuring Diffusion of Lipid-like Probes in Artificial and Natural Membranes by Raster Image Correlation Spectroscopy (RICS): Use of a Commercial Laser-Scanning Microscope with Analog Detection
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Measuring Diffusion of Lipid-like Probes in Artificial and Natural Membranes by Raster Image Correlation Spectroscopy (RICS): Use of a Commercial Laser-Scanning Microscope with Analog Detection

机译：通过光栅图像相关光谱法（RICS）测量人造膜和天然膜中类脂质探针的扩散：使用具有模拟检测功能的商业激光扫描显微镜

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摘要

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 (Dil-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.
机译：细胞膜内组成和相互作用的异质性转化为其组成成分的广泛扩散系数。因此，必须应用几种互补的微荧光技术，例如荧光相关光谱法（FCS），光漂白后的荧光回收率（FRAP）和单粒子跟踪（SPT），以探索膜组件的动力学。最近引入的光栅图像相关光谱法（RICS）提供的动态范围比分别使用这些方法的方法要宽得多，并且可以对动态属性进行空间映射。 RICS是在共聚焦激光扫描显微镜（CLSM）上实现的，通过在共聚焦图像的生成中利用扫描激光束携带的固有时间信息，可以实现较宽的动态范围。 RICS的最初介绍使用了两个光子激发和光子计数检测。但是，大多数CLSM系统都基于具有模拟检测功能的单光子激发。在这里，我们报告了这种商用CLSM（Zeiss LSM 510 META）在研究荧光脂质类似物1,1'-二十八烷基-3,3,3'，3'-四甲基-茚二碳菁高氯酸盐（在巨大的单层囊泡和活少突胶质细胞（即中枢神经系统中产生髓磷脂的细胞）的质膜中都存在Dil-C-18（5）。结果表明，带有模拟检测功能的商用CLSM上的RICS可以通过消除模拟检测系统引入的有害相关性，在膜扩散研究中获得可靠的结果。获得的结果与FRAP和FCS收集的结果相当。

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