Diffuse background DAPI staining disturps primary object identification

Hello,

Unfortunately, in just one of my wells out of 10 for my NIH3T3 experiment, I think a technical mistake of some sort was made with the DAPI. As you can see in the attached photos, there is this diffuse slight blue staining around the nuclei, that CellProfiler then mistakenly thinks is part of the nuclei. This gives a primary object identification of a way too big nuclei and it skews my whole analysis. Also it is not every single picture that has this problem, but some of them. I have also added a photo of how it ‘should’ look, from another well

Anyone have any idea how to fix this? I have tried the correctilluminationcalculate function (as seen in the unchecked modules of my pipeline) but all I get is a completely black image. Maybe I might be doing something wrong there.

Also I could erode the image, but it is not ideal for quantative image analysis since I am more of less deciding how big the nuclei are.

Pipeline JNO 2.7 edited for 202012008 best fit FIX E40QKO 2.cpproj (6.8 MB)

best fit NIH3T3 D1 E40Q 20X 06-Image Export-08_b0c0-3x0-1388y0-1040.tif (4.1 MB) best fit NIH3T3 D1 E40Q 20X 06-Image Export-08_b0c0x0-1388y0-1040.tif (4.1 MB)
best fit NIH3T3 D1 E40Q 20X 07-Image Export-09_b0c0-3x0-1388y0-1040.tif (4.1 MB) best fit NIH3T3 D1 E40Q 20X 07-Image Export-09_b0c0x0-1388y0-1040.tif (4.1 MB) best fit NIH3T3 D1 E40Q 20X 09-Image Export-11_b0c0-3x0-1388y0-1040.tif (4.1 MB) best fit NIH3T3 D1 E40Q 20X 09-Image Export-11_b0c0x0-1388y0-1040.tif (4.1 MB)
NIH3T3 D1 T37S 20X 03-Image Export-146_b0c0-3x0-1388y0-1040.tif (4.1 MB) NIH3T3 D1 T37S 20X 03-Image Export-146_b0c0x0-1388y0-1040.tif (4.1 MB)

Hi @EllenAppel,

I checked your pipeline & a sample image. I did two changes, one is use “RescaleIntensity” before Primaryobject module & other is change the Thresholding strategy & lower bound threshold value as shown in the screenshot. It looks like working.


Hope this helps.

Regards,
Lakshmi
www.wakoautomation.com

1 Like

Thank you so much Lakshmi!

This helps my project tremendously!

Hi again.

What setting did you use in rescale intensity?

Hi @EllenAppel,

You are welcome!! I used Rescaling method as “Stretch each image to use the full intensity range”.

Regards,
Lakshmi
www.wakoautomation.com