Colocalization

[Diamond]

Hello,

I have images that i am trying to use for quantification of colocalization.  However, i have some doubts and wanted to make sure that the method i am following is correct.  The method i am employing is as follows. 

1. Substract background by using IMageJ BG substraction from ROI. I use 3 as standard deviation as one of my channels has considerable background. 

2. Use ROI (freehand selection tool) to select individual cells.

3. Use Binary-convert to Mask to create masks individually for both the green and red channels for individual cells. I have three frames per experiment with each frame having around

    2-4 cells.

4. Use AND operation between masks and the original image for each channel individually to create ROIs with 0 background. This is done/cell.

5. Use JACoP plugin to measure Mander's Coefficients/cell using the "and" images for both the red and green channels. Done/cell.

Note:   1.  Both of my channels have different intensities with red being significantly lighter than the green one.

    2.  Had tried to use the images after ROI selection but have not been able to set a consistent threshold for the cells collected in different frames.

Any input would be appreciated.

Thanks in advance,

Cheers