Colocalization within Regions of Interest

Posted by Oleg Broytman on May 19, 2011; 12:44am
URL: http://imagej.273.s1.nabble.com/Colocalization-within-Regions-of-Interest-tp3684536.html

Greetings.  I need some advice about how to use Image J to carry out my colocalization analysis.  I am studying changes in protein localization in the Phrenic Motor Nucleus (PMN)- a cluster of neurons in the cervical spinal cord.  I have acquired Z-stack images under the confocal microscope of PMN neurons.  The PMN neurons are immunostained for Protein 1 on the Green channel of fluorescence and Protein 2 on the Red Channel of fluorescence.  They are also labeled with a tracer dye, the fluorescence of which is acquired on the Far Red channel.

What I would like to do is to analyze the extent of colocalization between Protein 1 and Protein 2 - not in the entire field, but only in the regions positively stained with the tracer dye.  So, I'd like to select regions of interest in each slice of the Z-stack in the Far Red channel (which would generate a whole list of ROIs for each stack), and then analyze Green/Red colocalization within each region of interest.

I could really use some advice on how to do this.  All the colocalization plugins I've encountered thus far analyze 2D colocalization in the entire 2D image, or 3D colocalization for the entire stack.  Is there a way to do colocalization only within a specific region of interest?

Thank you in advance for your help.

Sincerely,

Oleg Broytman
University of Wisconsin - Madison.