Measuring dapi intensity on neun positive cells

cellprofiler

#1

Hi
I am new to cell profiler, and having some trouble with my pipeline. I have some brain slides and i need to measure dapi intensities only in nuclei that are positive for neun staining. I would be greatfull if someone could tell me what is the best way to do that.

thanks in advance,
gal


#2

Hi,

From your brief description without example images, I can only imagine that:

  • If possible, identify the nuclei based on neun staining. Then measure the DAPI intensity inside the identified objects.

If neun objects are actually little dots, then:

  • you should first identify the nuclei objects based on DAPI staining
  • then measure both DAPI and neun intensity inside these identified objects, using MeasureObjectIntensity
  • then use FilterObject , take out the objects that have little-to-no neun. The remaining objects will be the ones that have DAPI signal.

Bests.


#3

One revision I’d make here- the DAPI measurements will not be “passed” onto the NeuN positive nuclei that survive the filter, so the workflow should be more like:

  • MeasureObjectIntensity for Nuclei in the NeuN channel
  • FilterObjects to filter the Nuclei into something like “NeuNPosNuclei”
  • MeasureObjectIntensity for NeuNPosNuclei in the DAPI channel

#4

Hi,
sorry for not being more specific and thanks for the help. My main problem is that the neun staining is very “dirty” and that’s why its harder to first identify the nuclei with the neun channel. So i’m thinking maybe the best way is to identify the nuclei with the dapi, than see which ones are also neun positive (green) and only measure their dapi intensity?
Only i am not so sure what is the way to recognize nuclei that are also neun positive after identifying with dapi. Should i recognize them using ID secondary objects? and once i do that, do they become merged, or i can measure the intensity only in the dapi channel and only in the cells that were found to be neun positive? should i first filter any cells?
I am attaching three pictures. ch1 is neun in green, ch3 is dapi in blue and ch4 merge.
I would really appreciate if i can get some help with this.
Thanks,
gal


#5

for some reason i am unable to upload the pictures. it starts uploading and than just stops. so hope someone can still help without the pictures


#6

Hi,

Yes, you can identify the nuclei with DAPI in IdentifyPrimaryObjects, then the protocol is what I described above

  • MeasureObjectIntensity for Nuclei in the NeuN channel
  • FilterObjects to filter the Nuclei into something like “NeuNPosNuclei” (you’ll have to play with what measurement makes sense, like MeanIntensity/MaxIntensity/IntegratedIntensity)
  • MeasureObjectIntensity for NeuNPosNuclei in the DAPI channel

#7

great, thank you very much!