Colocalization of fluorescent markers in confocal microscope images of plant cells
Colocalization of fluorescent markers in confocal microscope images of plant cells
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DOI：
10.1038/nprot.2008.31
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Pridmore, Tony P.
中科院分区：
文献类型：
--
作者：
French, Andrew P.;Mills, Steven;Pridmore, Tony P.
该协议描述了所需的步骤进行定量统计共定位的双色共聚焦图像，特别是植物细胞。该程序包括校准测试，以检查共焦显微镜的色度对准。提供软件工具来计算图像内的感兴趣区域上的Pearson和斯皮尔曼相关系数（“Pearson-斯皮尔曼相关共定位”ImageJ插件）。包括步骤，以帮助用户练习使用该软件。结果是图像中共定位量的定量估计。根据所需的细节，手动掩蔽每张图像大约需要1-15分钟，并且计算相关系数几乎是瞬时的。用于这种双色共定位的合适染料的实例包括俄勒冈州绿色或在绿色范围内的Alexa Fluor 488染料（用488-nm激光线激发）和在红色范围内的Alexa Fluor 555染料（用543-nm激光线激发）。
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).