Re: Ratiometric Analysis using Image J

Posted by Michael Cammer on Jul 17, 2007; 3:57am
URL: http://imagej.273.s1.nabble.com/Ratiometric-Analysis-using-Image-J-tp3698844p3698845.html

Essentially we simply background subtract, convert images to 32 bits and
divide the images.  Then we rescale from 0 to 2 (or something similar,
like 0.25 to 1.75) and use a LUT we lifted from Metamorph for display.

-Michael

> 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.
>

_________________________________________
Michael Cammer   http://www.aecom.yu.edu/aif/