I’m working on measuring the intensities of nucleoli. I’m doing fine identiying nuclei and nucleoli; you’ll notice a MaskObjects module in my pipeline designed to take out any odd particles that might have shown up as nucleoli that are not actually in nuclei. I’m aware that fluorescence varies naturally from coverslip to coverslip in a given experiment, even if all exposures are taken at the same length. To control for this natural variation, I compare nucleoli intensities to the intensities of surrounding nucleoplasm (which I call NENA in the pipeline for some reasons) with the idea that looking at these ratios should control for fluorescent variation since I’m just interested in changes in nucleolar intensity. However, when I look at these ratios, it seems as though CellProfiler does not tell me what my eyes tell me. For my “no damage” picture, it gives me a ratio of 1.08 for nucleoli:NENA, implying that the average pixel (I think?) intensity is greater in the nucleoli than in the nuclei. For the various other pictures I’ve included, either the ratio is again greater than 1 or only a bit below 1 when my eyes tell me it should be far below 1. I’m just not sure what is going on here. I’ve tried using Align to make sure my pictures were overlapping correctly, and found no difference in results. Do you have any suggestions?
I’m just sending the different channels for one experimental point, but I have more I can upload in separate responses if you wish. I’m also including the picture of the protein of interest; CellProfiler gives me a mean intensity ratio of .95 for mean nucleoli intensity to mean NENA (nucleoplasm) intensity. It looks like it should be less than that, right?
Thanks so much for your efforts on this program and website!
ku to send.mat (256 KB)