I am trying to use cell profiler to identify cells in a subregion of the retina. To do so, i am performing immunohistochemistry to stain my cells of interest that should positive for “islet1” antibody but negative for “sox2” antibody. I have written a pipeline for cell profiler but have encountered a few problems:
- I don’t seem to be able to preselect the retina subregion. I have found the option to select a rectangle or ellipse from the “crop” module but this doesn’t capture my area of interest which has an irregular shape that vary from one image to the next. Is there an option to draw/crop an arbitrary shape?
- I am trying to measure the islet1+/sox2- cells. To do so, I have used the module “relate” and then I have established a parental relationship between the two so that it counts islet1+/sox2- cells. To be able to export this in my image file on excel, I have used the module “filter by object measurement” but it doesn’t seem to count parental cells (islet1+) without children (sox2+) (nor parental cells with children). How should I fill this module to obtain automatically this cell count?
- using the module “identifyprimautomatic”, the cell count I get for islet1 seems quite accurate but the one I get for sox2 is not great (it seems that the background and the cells are not distinguished). It might be because there are usually very little sox2+ cells on my images. I have added a module “correct illumination” but it doesn’t seem to improve much. what do you suggest I do?
Thank you very much for your time.