I'm trying to measure the colocalization of two proteins in a .tiff image taken with a wide-field fluorescence microscope. In ImageJ I have split the image into the two channels (red and green), and now I have to apply a threshold, however when I threshold both channels the bright areas corresponding to the cells are located in the darkest part of the histogram instead of the brightest part, as you can see in this image for the red channel:
Why ImageJ is assigning darker values to the cells rather than to the background? I'm having problems with this as I cannot properly select these cells for the colocalization analysis. Anyone does have any clue? Any help would be greatly appreciated.