I am staining for proteins in the cell nucleus and would like some help with specific questions and the intuition behind when it is appropriate to normalize the single cell protein intensities to the single cell nuclear (DAPI/Hoescht) fluorescence. Either if someone can directly address the questions below or redirect me to some good reading material, that would be much appreciated.
When measuring the mean intensity per pixel in an object (cell), I understand that in addition to differences in the amounts of the molecule of interest, there are many other factors that can influence the intensity, including
- Changes in nuclear volume (e.g. from cell compaction).
- Cells at slightly different focal z-planes.
- Cells growing on top of each other so that their nuclei overlap and seem brighter than they are.
- Cells at different stages of cell cycle (at least this affects total DAPI)
Correctly accounting for these confounding factors would yield more accurate image quantification, and I have a few specific questions regarding how to go about this:
- Are these the most common confounding factors that are intended to be accounted for when normalizing protein to nuclear intensity?
- I have seen papers where they simply divide the intensity for the protein of interest with the nuclear intensity. Is there a good reference that shows that the nuclear and protein intensities co-varies linearly when the potentially confounding factors change?
- Are there instances when it is recommended to not normalize to nucelar content?
- To what degree does confocal slices get rid of the need for normalizing nuclei at different Z-position?
- Is the mean DNA intensity expected to be the same between all cells and not vary as the total DNA intensity does when cells progress through the cell cycle?
- If the system under study allows for it, would the best approach simply be to ensure that all cells are evenly spread out as a near perfect monolayer and avoid the need for any nuclear normalization?
As an example or the intuition, I include a couple of images.
The left shows nuclear intensity and the right shows a protein localized to the nucleus. The red ring indicates an area that is higher in nuclear intensity than the surrounding cells (not including the small, very bright dividing cells). The same area does not show any tendency of also having higher protein expression than the surrounding cells. I understand that the cells with the highest nuclear intensity will not necessarily be the cells with the highest protein intensity (then we wouldn’t need to measure both in the first place). However, in the dimmest cells expressing little or no protein, I would expect to see a weak variation in their intensity similar to the variation in nuclear intensity, which would suggested that the nuclear intensity is a good indicator of which cells are affected by confounding factors in the protein stain.
Thanks for reading my somewhat lengthy post.