I am facing problem with rescaling the intensity of my acquired images using Cell profiler. the pixels in the images are not visible and so downstream thresholding becomes a problem as I would like to use this to select my primary object. Also, my image contains tremendous amounts of clumped cells which I am able to segment using Otsu global and propagate method but the cells identified in the first image does not correspond exactly to the cells identified in the second image and so on. Also, there seems to be different amounts of cells identified from first image to the second so on. I have 24 time points corresponding to 24 images of the same field of cells and it is important to identify the same cells in each image to assess the change in intensity of fluorescence over time upon addition of ligand.
Thank you for your help.