yH2AX foci quantification

Hi everybody,

I want to quantify the phosphorylation of Histone yH2ax, I used the pipeline in your website, as my pictures are color I used the colortoGRay module, but then the indentifyprimobject module says that it has a memory error, and then the program stops working, could you tell me what I´m doing wrong? I sending a couple of pictures,

thank you very much


Hi Jonathan,

A couple suggestions first:
(1) See if you can export/save your images as grayscale. These images are not all blue or green, respectively, and are just color-coded for display anyway.
(2) Try and avoid JPG, as it is a “lossy” format. Try PNG or TIFF

The main settings to note when using color images is in NamesAndTypes – be sure to choose the Image Type = Color (not grayscale). You didn’t attach your pipeline, so I can’t be sure, but that is likely your problem. (What Example Pipeline of ours are you referring to?)

See my attached pipeline. I used the “Combine” option in ColorToGray, which is not optimal since the scale changes because it averages the RGB channels, but it is the simplest to do until you can export grayscale images directly.

Hope that helps,
DLpipe.cppipe (5.23 KB)

Hi David, Sorry for the delay,

Originaly I have the pictures in Tiff format, but they were too big to atach them, I’m ataching now the pipeline I modified from your collection could you check if it works? I will try to change the fomat to grayscale in other software,

Thank very much

Best regards

Adam.cpproj (605 KB)

HI Jonathan,

Are you saying that your pipeline doesn’t work? What are you requesting that we test? You are best to judge whether your pipeline is producing good results! If there is something erroring or you need to optimize some aspect of the pipeline, then by all means post detailed info here and we’ll take a look.
Note that you can upload your TIFFs to Dropbox or Google Drive and post the link here instead of uploading directly.

Glad you have access to TIFFS!


Hi David,

The pipeline transform the color pictures to gray, but when the identifyprimerobjects module starts to work the software stop working I´m ataching some screen captures of the process,

Best regards


If you’re still having trouble, can you attach your pipeline here? Sorry for the delay.