title: Colocalization of fluorescent markers in confocal microscope images of plant cells.
abstract: 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).
authors.affiliation: Centre for Plant Integrative Biology, Main Building, University of Nottingham, Sutton Bonington, LE12 5RD, UK. andrew.french@cpib.ac.uk
authors.forename: Andrew P
authors.initials: AP
authors.lastname: French
authors.forename: Steven
authors.initials: S
authors.lastname: Mills
authors.forename: Ranjan
authors.initials: R
authors.lastname: Swarup
authors.forename: Malcolm J
authors.initials: MJ
authors.lastname: Bennett
authors.forename: Tony P
authors.initials: TP
authors.lastname: Pridmore
mesh_terms: D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D059828:Plant Cells; D010940:Plant Proteins / Q000378:metabolism*; D021381:Protein Transport; D012984:Software
chemical_list: D005456:Fluorescent Dyes; D010940:Plant Proteins