The problem I am trying to explain (and I promise I won’t insist on this anymore ) is that the colour LUT will not tell you anything about the original scan intensity data (i.e. If you plan to execute all this in a batch macro, you might want to close all open images and clear the ROI Manager before starting the processing for each image: close("*") So when you then select the first entry in the ROI Manager, it will be the selection you want, which will be shown on your original image. When you then close the black and white image (I decided to call it “Mask” in the “duplicate” command), your original Image will again be the next available image. From this image you create the selection as I described above and add it to the ROI Manager - like “depositing” the selection. Then you insert the code that is used by the color thresholder to create the black and white image. So, after opening the image, you execute: run("Duplicate.", "title=Mask") To do that, you have to “duplicate” your original image, before processing it to create the black and white “mask”, from where you get the selection. Let me know if this works or if you have any more questions! And maybe, if this solves your initial question and you can - for now - analyze your images with the raw data you have available right now, you can come back for some very useful insight into raw data quality, what LUTs are, what intensity values mean, and how information about intensity gets lost in the RGB images you use at the moment. You can select the positive signal by subsequently using: setThreshold(1,255) When you execute this code, the result is a black and white image, defining your background and positive signal.Ĩe19ca2f54262e891a966191acabf2608b35b5e3-1.tif (103.4 KB) ImageCalculator("AND create", "Result of 0","2") Autogenerated macro, single images only! You say you already know how to get the macro code for the colour thresholding - it looks something like this, I assume? // Color Thresholder 2.3.0/1.53q Dear points out the flaws of your raw data (and you should try to address them in the future), I wanted to quickly give you a solution to your original question:
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