AUTOMATED DETECTION AND TRACKING OF INDIVIDUAL AND CLUSTERED CELL-SURFACE LOW-DENSITY-LIPOPROTEIN RECEPTOR MOLECULES
AUTOMATED DETECTION AND TRACKING OF INDIVIDUAL AND CLUSTERED CELL-SURFACE LOW-DENSITY-LIPOPROTEIN RECEPTOR MOLECULES
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DOI：
10.1016/s0006-3495(94)80939-7
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发表时间：
1994-05-01
影响因子：
3.4
通讯作者：
WEBB, WW
中科院分区：
文献类型：
--
作者：
GHOSH, RN;WEBB, WW
我们开发了一种检测、识别和跟踪人类皮肤成纤维细胞表面每一个单独的低密度脂蛋白受体（LDL-R）分子和小受体簇的技术。分子识别和细胞表面所有个体受体的高精度（30 nm）同步自动跟踪利用定量延时低光数字视频荧光显微镜，通过在图像处理工作站执行的专用算法进行分析。LDL- rs用具有生物活性的荧光LDL衍生物dl -LDL标记。通过测量图像中亚分辨率荧光点辐射的荧光功率来识别单个LDL-Rs和未解析的小粉尘；单颗粒的鉴定是由四个独立的技术确定。开发了一种自动跟踪程序，可以同时跟踪，无需用户干预，通过数百个延时图像帧的序列跟踪众多荧光颗粒。发现跟踪精度的限制取决于被跟踪粒子图像的信噪比和显微镜系统的机械漂移。我们描述了涉及到的方法(i)延时获取低光图像，（ii）荧光衍射限制点图像的同步自动跟踪，（iii）高精度和局限性的粒子定位，以及（iv）检测和识别单个和集群LDL-Rs。这些方法普遍适用，为可视化和测量活细胞表面单个整体膜蛋白的动力学和相互作用提供了有力的工具。
We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small dusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, acid without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces.