I divided the image into four classes: background, the dark area in the center ("blob"), the cells themselves ("class 1") and the median line running through each cell ("class 2"). I played a bit with Trainable Weka Segmentation and got some potentially promising, but still-not-yet-good-enough, results: In your case, I would suggest giving the former a try. So far, your attempts fall more into the latter category. The Fiji wiki page on Segmentation discusses two primary ways of approaching image segmentation: the Trainable Weka Segmentation plugin, and a more flexible macro-based workflow. Overlapping objects can be a tough problem. Pleas help (either with the first or second code!) Thanks! :) Run("Auto Threshold", "method=Otsu white") ImageCalculator("Subtract create", a,"_seed") Run("GreyscaleReconstruct ", "mask="+a+" seed=_seed create") I also tried using GreyscaleReconstruct but I was not that successful either. I am using the macros on a stack of images. Run("Analyze Particles.", "size=200-2000 circularity=0.50-1.00 show= display exclude clear summarize add in_situ") Run("Auto Threshold", "method=Yen white stack") Run("Subtract Background.", "rolling=5 light sliding stack") However, I have a hard time removing the overlap between cells and for the program to distinguish between the clumps. I have been trying to make a macros for counting the cells in the image.
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