French Andrew P, Mills Steven, Swarup Ranjan, Bennett Malcolm J, Pridmore Tony P
Centre for Plant Integrative Biology, Main Building, University of Nottingham, Sutton Bonington, LE12 5RD, UK.
Nat Protoc. 2008;3(4):619-28. doi: 10.1038/nprot.2008.31.
This protocol describes the steps needed to perform quantitative statistical colocalization on two-color confocal images, specifically of plant cells. The procedure includes a calibration test to check the chromatic alignment of the confocal microscope. A software tool is provided to calculate the Pearson and Spearman correlation coefficients ('Pearson-Spearman correlation colocalization' ImageJ plug-in) across regions of interest within the image. Steps are included to help the user practice using the software. The result is a quantitative estimate of the amount of colocalization in the images. Manual masking takes about 1-15 min per image, depending on the detail required, and calculating the correlation coefficients is almost instantaneous. Examples of suitable dyes for such two-color colocalization include Oregon Green or Alexa Fluor 488 dyes in the green range (excited with 488-nm laser line) and Alexa Fluor 555 dye in the red range (excited with 543-nm laser line).
本方案描述了对双色共聚焦图像（特别是植物细胞图像）进行定量统计共定位所需的步骤。该程序包括一个校准测试，以检查共聚焦显微镜的色差校准。提供了一个软件工具来计算图像中感兴趣区域的皮尔逊和斯皮尔曼相关系数（“皮尔逊 - 斯皮尔曼相关共定位”ImageJ插件）。其中包含帮助用户练习使用该软件的步骤。结果是对图像中共定位量的定量估计。根据所需细节，手动掩膜每张图像大约需要1 - 15分钟，而计算相关系数几乎是瞬间完成的。适用于这种双色共定位的染料示例包括绿色范围内的俄勒冈绿或Alexa Fluor 488染料（用488 nm激光线激发）以及红色范围内的Alexa Fluor 555染料（用543 nm激光线激发）。
