Colocalization of fluorescent markers in confocal microscope images of plant cells

被引：312

作者：

French, Andrew P. ^{[1
]}

Mills, Steven ^{[1
,2
]}

Swarup, Ranjan ^{[1
]}

Bennett, Malcolm J. ^{[1
]}

Pridmore, Tony P. ^{[1
,2
]}

机构：

[1] Univ Nottingham, Ctr Plant Integrat Biol, Loughborough LE12 5RD, Leics, England

[2] Univ Nottingham, Sch Comp Sci, Nottingham NG8 1BB, England

来源：

NATURE PROTOCOLS | 2008年 / 3卷 / 04期

基金：

英国生物技术与生命科学研究理事会;

关键词：

D O I：

10.1038/nprot.2008.31

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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).

引用

页码：619 / 628

页数：10

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