I’m helping a colleague to develop a pipeline to segment human cells with fluorescent anti-body staining (abbrv. FAB). The cells are also stained with DAPI to identify the nuclei. However, I’m having a really hard time identifying cells due to the large variation in number of cells present in each site, as well as the uneven illumination in each image.
I’ve uploaded some sample images here: dropbox.com/sh/1qksoc2bqbgb … C6RS2iHRHa
The pipeline I’ve tried is pretty simple (see attached): I correct the illumination of the DNA channel -> identify primary for nuclei -> correct illumination of the GFP channel -> identify secondary for cells based on nuclei -> overlay outlines and save to tif.
My tests show that I’m hugely over-segmenting. Many objects do not contain cells, both in those images that are mostly empty of cells and in those with plenty of cells. I’ve tried varying the segmentation method as well as the parameters therein, but still no improvement. Am I not doing enough to pre-process the images? Or have I not set up my segmentation modules correctly? I’d appreciate any tips that anyone can provide.
overlay_seg_jeff_test.cpproj (1.27 MB)