Pompey Shanica N, Michaely Peter, Luby-Phelps Katherine
Department of Cell Biology, UT Southwestern Medical School, Dallas, TX, USA.
Methods Mol Biol. 2013;1008:439-53. doi: 10.1007/978-1-62703-398-5_16.
Fluorescence microscopy can be used to assess quantitatively the interaction between a ligand and its receptor, between two macromolecules, or between a macromolecule and a particular intracellular compartment by co-localization analysis. In general, this analysis involves tagging potential interacting partners with distinct fluorophores-by direct labeling of a small ligand, by expression of fluorescent cDNA constructs, by immunofluorescence labeling, or by some combination of these methods. Pairwise comparison of the fluorescence intensity of the two fluorophores at each pixel in a two channel digital image of the sample reveals regions where both are present. With appropriate protocols, the image data can be interpreted to indicate where the potential interacting partners are co-localized. Keeping in mind the limited resolution of the light microscope, co-localization is often used to support the claim that two molecules are interacting.All quantitative methods for evaluating co-localization begin with identifying the pixels where the intensities of both color channels are above background. Typically this involves two sequential image segmentation steps: the first to exclude pixels where neither channel is above background, and the second to set overlap thresholds that exclude pixels where only one color channel is present. Following segmentation, various quantitative measures can be computed to describe the remaining subset of pixels where the two color channels overlap. These metrics range from simple calculation of the fraction of pixels where overlap occurs to more sophisticated image correlation metrics. Additional constraints may be employed to distinguish true co-localization from random overlap. Finally, an image map showing only the co-localized pixels may be displayed as an additional image channel in order to visualize the spatial distribution of co-localized pixels. Several commercial and open source software solutions provide this type of co-localization analysis, making image segmentation and calculation of metrics relatively straightforward. As an example, we provide a protocol for the time-dependent co-localization of fluorescently tagged lipoproteins with LDL receptor (LDLR) and with the early endosome marker EEA1.
荧光显微镜可用于通过共定位分析定量评估配体与其受体之间、两个大分子之间或大分子与特定细胞内区室之间的相互作用。一般来说，这种分析包括用不同的荧光团标记潜在的相互作用伙伴——通过直接标记小配体、通过表达荧光cDNA构建体、通过免疫荧光标记或通过这些方法的某种组合。在样品的双通道数字图像中，对每个像素处两种荧光团的荧光强度进行成对比较，可揭示两种荧光团都存在的区域。通过适当的方案，图像数据可以被解释为指示潜在的相互作用伙伴共定位的位置。考虑到光学显微镜分辨率有限，共定位常被用于支持两个分子相互作用的说法。所有评估共定位的定量方法都始于识别两个颜色通道强度均高于背景的像素。通常这涉及两个连续的图像分割步骤：第一步排除两个通道强度均未高于背景的像素，第二步设置重叠阈值以排除仅存在一个颜色通道的像素。分割后，可以计算各种定量指标来描述两个颜色通道重叠的剩余像素子集。这些指标范围从简单计算重叠像素的比例到更复杂的图像相关指标。可能会采用额外的约束条件来区分真正的共定位和随机重叠。最后，可以将仅显示共定位像素的图像地图作为额外的图像通道显示，以便可视化共定位像素的空间分布。有几种商业和开源软件解决方案提供这种类型的共定位分析，使得图像分割和指标计算相对简单。例如，我们提供了一个关于荧光标记的脂蛋白与低密度脂蛋白受体（LDLR）以及早期内体标记物EEA1随时间共定位的方案。
