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