Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera. Low-density lipoprotein and influenza virus receptor mobility at 4 degrees C.
Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera. Low-density lipoprotein and influenza virus receptor mobility at 4 degrees C.
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发表时间：
1992-02
影响因子：
4
通讯作者：
C. M. Anderson;G. Georgiou;I. Morrison;G. V. Stevenson;R. Cherry
中科院分区：
文献类型：
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作者：
C. M. Anderson;G. Georgiou;I. Morrison;G. V. Stevenson;R. Cherry
本文介绍了一种基于冷却慢扫描CCD摄像机的荧光成像系统，用于跟踪活细胞表面的受体。该技术适用于可以用几十个荧光团标记的颗粒受体，如脂蛋白和病毒。通过将荧光分布拟合到二维高斯函数来确定每个图像中的单个颗粒的位置在25 nm内。该过程还提供了强度的精确测量，其用作从帧到帧自动跟踪粒子的标记。该方法适用于研究4 ℃下人皮肤成纤维细胞上LDL和流感病毒颗粒受体的迁移率。与FRAP（光漂白后的荧光恢复）以前的研究相反，发现受体在4 ℃下具有低但可测量的迁移率。个别粒子轨迹的分析表明，虽然一些受体进行随机扩散，其他人进行定向运动（流动）或扩散限制到一个域。提出了一个程序细分受体根据其不同类型的运动，从而确定其运动参数。受体在4 ℃下不完全固定的发现对于在该温度下进行的受体分布的研究是重要的。
A fluorescence imaging system, based on using a cooled slow-scan CCD camera, has been developed for tracking receptors on the surfaces of living cells. The technique is applicable to receptors for particles such as lipoproteins and viruses that can be labeled with a few tens of fluorophores. The positions of single particles in each image are determined to within 25 nm by fitting the fluorescence distribution to a two-dimensional Gaussian function. This procedure also provides an accurate measure of intensity, which is used as a tag for automated tracking of particles from frame to frame. The method is applied to an investigation of the mobility of receptors for LDL and influenza virus particles on human dermal fibroblasts at 4 degrees C. In contrast to previous studies by FRAP (fluorescence recovery after photo-bleaching), it is found that receptors have a low but measurable mobility at 4 degrees C. Analysis of individual particle tracks indicates that whilst some receptors undergo random diffusion, others undergo directed motion (flow) or diffusion restricted to a domain. A procedure is proposed for subdividing receptors according to their different types of motion and hence determining their motional parameters. The finding that receptors are not completely immobilised at 4 degrees C is significant for studies of receptor distributions performed at this temperature.