Analysis of Signaling Events by Combining High-Throughput Screening Technology with Computer-Based Image Analysis
Analysis of Signaling Events by Combining High-Throughput Screening Technology with Computer-Based Image Analysis
复制标题
DOI：
10.1126/scisignal.137pl2
复制
发表时间：
2008-09-16
影响因子：
7.3
通讯作者：
Stochaj, Ursula
中科院分区：
文献类型：
--
作者：
Kodiha, Mohamed;Brown, Claire M.;Stochaj, Ursula
细胞内的信号传导和细胞间的通讯依赖于众多信号传导事件的协调，而这种大量的信息流必须在空间和时间上得到适当的组织。所有这些过程和最终细胞反应的共同和关键是信号组分及其靶标的正确空间分布。这个基本概念适用于大量的信令过程。通常重要的是量化不同细胞区室中信号分子的定位，以检测微妙的变化或定义触发后续事件所需的特定位置中信号分子的阈值水平。特别重要的是核和细胞质事件的分离，需要灵敏的方法来测量它们对信号转导的贡献。这里描述的程序允许位于细胞核，细胞质，或在核膜的荧光信号的定量。这些方法依赖于高通量成像设备、共聚焦显微镜和测量感兴趣的隔室中的荧光强度的软件模块。我们讨论了选择适当的图像采集设备和适当的软件模块来量化不同细胞室中的荧光的基本原理。最初，为多孔板开发了用于高速图像采集的高通量技术。我们采用高通量技术对盖玻片上生长的细胞进行图像采集。通过共聚焦显微镜获得沿z轴沿着的更高空间分辨率的图像。对于后续分析，选择适当的软件模块对于快速可靠地定量荧光强度至关重要。
Intracellular signaling and cell-to-cell communication depend on the coordination of numerous signaling events, and this large flow of information has to be properly organized in space and time. Common and critical to all of these processes and the ultimate cellular response is the correct spatial distribution of signaling components and their targets. This fundamental concept applies to a large number of signaling processes. It is frequently important to quantify the localization of signaling molecules within different cellular compartments to detect subtle changes or to define threshold levels of signaling molecules in a certain location that are necessary to trigger subsequent events. Of particular importance is the separation of nuclear and cytoplasmic events, and sensitive methods are required to measure their contribution to signal transduction. Procedures described here allow the quantification of fluorescence signals located in the nucleus, cytoplasm, or at the nuclear envelope. The methods rely on high-throughput imaging equipment, confocal microscopy, and software modules that measure the fluorescence intensity in the compartment of interest. We discuss the rationale for selecting the appropriate equipment for image acquisition and the proper software modules to quantify fluorescence in distinct cellular compartments. Initially, high-throughput technology for high-speed image acquisition was developed for multiwell plates. We adapted high-throughput technology for image acquisition for cells grown on cover slips. Images of higher spatial resolution along the z axis were acquired by confocal microscopy. For subsequent analyses, the choice of appropriate software modules is critical for rapid and reliable quantification of fluorescence intensities.