Ratiometric Analysis using Image J

Posted by Sam Frankel on Jul 17, 2007; 3:30am
URL: http://imagej.273.s1.nabble.com/Ratiometric-Analysis-using-Image-J-tp3698844.html

Hello,

I'm working on a project attempting to use ratiometric staining of
cells in culture to analyze the cytoskeleton.  I've been through a
couple of web sites reading about
ImageJ and ratiometric imaging, but the instructions just don't seem
to be pointing me in the right direction and I was wondering if anyone
could help.

My field test is to stain cells with DNAseI (red channel) to label
globular actin
(G-Actin) and Alexa 488 Phalloidin (green channel) to label
polymerized actin (F-Actin). For each cell, I take a photograph of the
green channel and a photograph of the red channel using an
epifluorescent microscope. I am trying to figure out a way to get a
ratio of red G-Actin labeling to green F-actin labeling in selected
regions of interest (after subtracting out the background from each
image). Ideally I'm looking for a relatively simple way to compare
pixel intensities from certain selected areas.

Can anyone get me started or point me in the right direction? I've
been doing some experimenting with imageJ but could use a hand.

Thanks very much for your time,

-Sam F.