I have a pipeline that works quite well at defining the nuclei and the cytoplasm in order to quantify the levels of transcription factors in the two areas.
Even though most pictures are quite comparable in terms of background and are accurately interpreted by the pipeline (example in file A), my most recent data contains some files that confuses the pipeline leading it to draw huge cytoplasm. The picture eventually looks like patchwork!
My IdentifySecondaryObjects module is Global threshold strategy and Otsu. I have tried the three classes (example in file B) and the two classes (example in file C) and it's not getting better. Increasing the threshold to the point where the cytoplasm is accurately interpreted results in too stringent delineation.
Would you have any pointers on how to deal with this issue?
Many thanks in advance for your support!