So I am trying to measure the intensity of the bands and before I would just try to crop each band around the same size. However I want to be more precise so I want CellProfiler to see each band as an object and give the intensity of each band. I have come close however I can’t seem to get CellProfiler not to split some of the bands into 2 separate objects. I have changed the thresholding method and the diameter of objects to try and get each band as 1 object however I can’t seem to get all of them to work. Here is my latest attempt.
TESTSTAT3-Western Blot Quantification - Advance - ej copy.cp (7.54 KB)
If your bands are well-separated, you could turn off declumping altogether: Change “Method to distinguish clumped objects” to “None.” That would gaurantee that the individual objects are joined.
If you have images where the bands are touching, I would suggest instead unchecking the “Automatically calculate minimum allowed distance between local maxima” and entering a larger number, perhaps half the length of the band in pixels, and also manually increasing the smoothing filter size. A smoothing filter size of 20 and a minimum maxima distance of 30 give the same result as turning off declumping.
THANKS!! it worked