Ratiometric Analysis using Image J

[Sam Frankel]

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]

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/

[B. Prabhakar Pandian]

Hello,
          I have multiple images in which I want to create a circle with
200µm diameter with a varying specified point as the center of the
circle. Is there a way  I can do this.
I am a beginner in ImageJ so any suggestions will help me a lot.

Thanks,

-Prabhakar

[Wayne Rasband]

Here is a macro tool that draws circles. Double click on the tool to
change the circle diameter. Double click on the eye dropper tool to
change the color used to draw the circles. Use the Analyze>Set Scale
command to define the unit and scale. Add the code to
ImageJ/macros/StartupMacros.txt to have the tool added to the tool bar
when ImageJ starts.

-wayne

     var diameter = 200;
     macro "Circle Tool - C00cO11cc" {
         getCursorLoc(x, y, z, flags);
         getPixelSize(unit, w, h);
         d = diameter;
         drawOval(x-d/(w*2), y-d/(h*2), d/w, d/h);
     }
     macro "Circle Tool Options" {
         getPixelSize(unit, w, h);
         diameter = getNumber("Diameter ("+unit+"): ", diameter);
     }


On Jul 17, 2007, at 12:45 PM, B. Prabhakar Pandian wrote:

> Hello,
>          I have multiple images in which I want to create a circle
> with 200µm diameter with a varying specified point as the center of
> the circle. Is there a way  I can do this.
> I am a beginner in ImageJ so any suggestions will help me a lot.
>
> Thanks,
>
> -Prabhakar
>