I been trying for a while now to segment some cells that are quite dim due to a depolymeriser treatment. (These cells are part of an assay that will include a range of compounds including depolymerisers and stabilizers). IdentifyPrimAutomatic works fine on the nuclear stain … but I have difficulty in getting a good (+consistent) result with IdentifySecondary. I have experimented with many settings. Typically some cells in the image might be segmented well, but then others have greatly enhanced boundaries.
So I was wondering if I might be able to get any further advice or suggestions. Any help or comments would be most welcome. I have attached some sample images (each with a nuclear and secondary stain). Note that although the images are dim, the cell bodies do become visually apparent if the contrast is increased greatly.
PS I have tried all the different algorithms, and experimented with threshold correction factor, lower/upper bounds, adaptive vs. global etc., and with “per object” methods. Also the images have already been illumination corrected.
Thank you (+ thanks also for making cellprofiler available to us),