I am trying to use Cell Profiler on staining of human tissue sections. The tissue has multiple different cell types with different shapes in close proximity, and each cell type has a separate surface marker. In addition to analysis of individual cell types, I would like to create a mask for the entire tissue that accurately separates cell borders (hopefully remaining faithful to different cell shapes), but I was having trouble.
One thing I have been trying is to create multiple different “sub-masks” and merging them, with the following process:
- I start with primary objects for the whole image, as all the nuclear staining is the same.
- I then identify and mask secondary objects from each different surface marker.
- I would then filter each mask for positivity of that given marker to create a subset of the mask. (For example, I would mask secondary objects based on marker A and then filter out only the cells positive for marker A, giving a “sub-mask” of only cell type A).
- Finally, I merge these different submasks in a different software – haven’t figured out how to do this within CellProfiler yet. (If it can be done in CellProfiler, please let me know!)
While this method seemed promising, I have run into a lot of issues with faithful segmentation since the different cell types are in close proximity to one another. For example, when I identify secondary objects using marker A, the program is not “aware” of any other membrane markers (say, marker B). Therefore, when identifying these secondary objects, it may extend the membrane to include a region with marker B, and I will now get a false double-positive cell.
Please let me know if there is a good method to get faithful segmentation and masking of differing cell types with different markers that are in close proximity within a tissue.
Thanks for your help!