Colocalization of fluorescent markers in confocal microscope images of plant cells

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescent Dyes
  • Microscopy, Confocal*
  • Microscopy, Fluorescence / methods*
  • Plant Cells*
  • Plant Proteins / metabolism*
  • Protein Transport
  • Software


  • Fluorescent Dyes
  • Plant Proteins