Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes.
Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes.
复制标题
成像荧光相关光谱：平面膜上 IgE 分布不均匀。
DOI：
10.1016/s0006-3495(96)79766-7
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Thompson,NL
中科院分区：
文献类型：
--
作者：
Huang,Z;Thompson,NL
荧光相关光谱可用于检测和表征尺寸小于或近似等于光学分辨率的分子簇。在这里，我们报告的方法，其中从一个单一的荧光图像的像素到像素的荧光波动空间自相关的发展。在这些测量中，四甲基罗丹明标记的，抗三硝基苯基IgE抗体特异性结合到基板支持的平面膜组成的三硝基苯基-氨基己酰基二棕榈酰磷脂酰乙醇胺和二棕榈酰磷脂酰胆碱。用来自全内反射激光束的渐逝场照射抗体包被的膜，并用冷却的CCD照相机记录由IgE包被的膜产生的荧光。在背景减除之后，针对渐逝照明的椭圆高斯形状校正图像。所得到的图像的空间自相关函数产生两个有用的参数：外推的初始值，这是有关的平均集群强度和密度;和相关距离，这是有关的平均集群大小。这些参数随IgE密度而变化，未标记的多克隆抗IgE增强了IgE分布的不均匀性。从含有荧光标记的脂质，而不是结合，标记的IgE的平面膜的图像计算的自相关函数表明，空间不均匀性是突出的，只有在IgE的存在下。用荧光微珠来演示原理和方法。
Fluorescence correlation spectroscopy is useful for detecting and characterizing molecular clusters that are smaller than or approximately equal to optical resolution in size. Here, we report the development of an approach in which the pixel-to-pixel fluorescence fluctuations from a single fluorescence image are spatially autocorrelated. In these measurements, tetramethylrhodamine-labeled, anti-trinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenyl-aminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The antibody-coated membranes were illuminated with the evanescent field from a totally internally reflected laser beam, and the fluorescence arising from the IgE-coated membranes was recorded with a cooled CCD camera. The image was corrected for the elliptical Gaussian shape of the evanescent illumination after background subtraction. The spatial autocorrelation functions of the resulting images generated two useful parameters: the extrapolated initial values, which were related to the average cluster intensity and density; and the correlation distances, which were related to the average cluster size. These parameters varied with the IgE density, and unlabeled polyclonal anti-IgE enhanced the nonuniform IgE distributions. The autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Fluorescent beads were used to demonstrate the principles and the methods.