Why DAPI (nuclei staining) is present in Cytoplasm?


#1

Dear Team,

This is a very unusual thing that I have observed.
We are using DAPI as nuclear staining and two other proteins with different dyes.

Upon running CP, when I looked at total integrated Intensity for blue (DAPI) in nucleus & propcytoplasm, i found that this intensity is not only present in propcytoplasm but also, its much higher than the nucleus.

Its a serious concern for the lab. What could be the reason?
Plz help us and let me know for any information required !!

Thanks
Mridul KK


#2

Hi,

One reason for this is that the integrated intensity is the sum of the fluorescent intensities of all pixels within the object. If your “propcytoplasm” object includes the cytoplasm plus the nucleus (as opposed to a tertiary object which is the cell without the nucleus), this integrated intensity will include that of both of the nucleus and the cytoplasm. So it is not surprising that the integrated intensity will be higher than that of the nucleus in this circumstance; indeed, it is guaranteed to be the case.

If you are interested in the integrated intensity of the cytoplasm only (without the nucleus), use the IdentifyTertiary module. If this is already what you are doing, then are two possibilities:

  • There is background staining of DAPI in the images, which is not unusual. In this case, there is going to be some baseline amount of DAPI intensity in the cyctoplasm.

  • Again, since the integrated intensity is the sum of the pixel intensities, if the cytoplasm is much larger than the nucleus, the intensity sum in the cytoplasm may just simply add up to a larger number than in the nucleus because there are more pixels in the cytoplasm to sum over.

Regards,
-Mark