I have a set of cell images which have very bright intensity on the membrane, weak intensity in the cytosol, and very weak intensity in the extracellular space. I’m planning on doing a set of image analysis steps in CellProfiler, the first of which is thresholding out the signal so as to separate the membrane/cytoplasm from the extracellular background fluorescence. Judging by the pixel intensity values that I see by just scanning the cursor around the image, this should be possible - the weak cytoplasmic fluorescence is still ~2x greater than the extracellular signal. I’m playing with different thresholds and I’d like to validate the results by showing the pixels that get cut off: that is, I apply a low pass filter so that everything above threshold goes away (becomes zero) and everything below threshold retains its associated greyscale value.
I now want to make the image binary so that I can see exactly which pixels get thresholded out. However, when I perform Process->Binary->Make Binary, I see that ImageJ again performs some sort of thresholding - all but a few speckles in the extracellular space get set to zero, when instead they should have been set to 255 (Because they were low non-zero). Is there a way to simply highlight all non-zero pixels and make them the same color/intensity?
Thanks for any help.