I’m working with fibroblast cells, staining with DAPI and a tubulin antibody that fluoresces green. We are trying to identify changes in morphology as a result of a change in the material the cells are cultured on. Another graduate student and I worked to develop our original pipeline (attached) that seemed to work very well the first time around. This time, I am having issues where there are essentially no primary objects identified, which results in no secondary objects and no results at all. When I have the pipeline show me what it has identified primary objects, it just shows a completely blue image and no objects identified or outlined. I’ve started to think maybe there is an issue with my images being too bright, and that I haven’t changed the right settings in my pipeline to compensate for that. Other than that I don’t have any ideas at the moment, because like I said we had been getting very accurate identifications and seemingly reasonable results.
I’ve attached a blue and green fluorescent image, what CellProfiler shows me after the conversion of these images to gray scale, as well as the results that show after CellProfiler attempts to identify primary and secondary objects. Maybe this is more than you need, but I wanted to give as much information as possible.
Please let me know if you need any more info from me, and thanks in advance for your input!
secondary objects.pdf (125 KB)
primary objects.pdf (45.8 KB)
color to gray green.pdf (83.9 KB)
colortogray blue.pdf (36.9 KB)
PLR Stain Analysis CP pipeline.cp (25.2 KB)