Normalize Intensities two different sample stacks using DAPI as reference?

Thank you in advance for anyone checking this out! :slightly_smiling_face:

The .rar file in the link contains two 3-channel confocal stacks each of two different cell types.
The 3 channels are DAPI, Green and Red. Both images were captured with the exact same settings. The Red and Green channels are expected to change, because of know cell differences for the proteins. However, DAPI was captured at lower intensity in #8 (I don’t know why). It is expected that they it would be similar in both since they were stained the same. Or not? Given what was visible by eye, this difference is unexpected.
Comapring file #1 with file #8 one can see that the intensities of all channels are lower in #8. But looking at DAPI one could argue that the lower channel intensities is a general effect and not ‘protein level’ specific.

To see if this can be studied I want to know if I can use the DAPI ratios in both samples to ‘equalize’ intensities, and then use that to normalize the other channels across both samples. Is that a true quantitative effect?

Comments/suggestions welcome, especially if this is not the right thing to do!!

From my experience DAPI staining is terribly inconsistent between samples, however I have not really done the staining myself, only observed it in other people’s samples. It is the one channel I have occasionally been fine with altering at acquisition time.

Anecdotally, it seems like samples with fewer cells are generally brighter, especially in DAPI stained mounting media. I suspect that is due to there only being a limited amount of DAPI within diffusion range of X number of nuclei, but have not tested anything.

On the other hand, there are plenty of experimental/physical reasons for all channels to be dimmer in a given sample, so different staining intensity in DAPI can mean something, it just doesn’t have to. Distance from the coverslip, for instance, can have an impact. Or if samples were prepared on the slide instead of on the coverslip, the depth of mounting media added at that location.

There are also biological/biochemical reasons for DAPI to be brighter or dimmer, depending on how condensed the chromatin is, for example. Probably plenty of other things I am missing as well :slight_smile:

*these are all just ideas I am throwing out there, did not actually take a look at your specific example.