Hi! I’m creating a pipeline to quantify transfection efficiency by comparing the number of GFP+ to total cells (DAPI). The outline of my pipeline is set up, but I’m having trouble getting my segmentation accurate and robust enough for general use.
In brief, I count DAPI and GFP cells with IdentifyPrimaryObject modules, relate the two with nuclei as the child, then filter out nuclei without GFP+ parents. However, my segmentation of both DAPI and GFP+ cells isn’t always accurate/consistent across different images (especially since the percentage and intensity of GFP+ cells can vary greatly between images).
Any advice about settings in IdentifyPrimaryObjects for DAPI and GFP+ cells, or other modules that could help, would be greatly appreciated! Thanks!
TransfectionForum_DAPI1_GFP1_10x.cpproj ! (84.9 KB)
Image set that works well
Counts DAPI well, ‘oversegments’ GFP:
Counts DAPI well, ‘undercounts’ GFP (there are faint cells it doesn’t count. This sometimes happens with DAPI as well)