I am working (first time) with Cell Profiler on a series of confocal images, showing MSCs on gelatin hydrogel discs. I am trying to tweak the pipeline in order to identify the nuclei in the blue channel and, depending if there is cytoplasmatic marker expression in the green/red channel (no matter how strong or weak), to count how many positive cells I have got.
The problem is, I am having a hard time to get rid of the strong background (the gelatin here has high autofluorescence), which ends up being included in CP analysis, incuding “nuclei” or “marker expression” where in reality there’s none. Additionally, I cannot get a good segmentation when the nuclei are too close to each other, and I don’t really know how to adjust the settings to define them properly.
The results I am getting of course have very high variability, and although I tried different approaches to reduce as much as possible the autofluorescence or to get clear images, these are the best I can get.
I can provide more examples if needed, and I hope anyone can give me some suggestions or advices on how to proceed.
Thank you all.