For the above histogram, what you are seeing are the pixel intensities for every pixel on the left panel of the movie. This means that you are measuring the intensity of not only the fluorophore spots that you are interested in, but also the background. Ideally, you will select a minimum intensity threshold in your PSM file that allows you to ignore the intensity from the background, although it is not always possible to do this cleanly. If you look at the above histogram, you can see that there are three distinct peaks. I assume that the peak that is farthest to the left is from the background intensity, whereas I assume that the peak that is farthest to the left is from the fluorophore spot intensity. I am not sure what the middle peak is from, so I set the minimum intensity value in my PSM file to be roughly 1800. This is largely empirical, though. In general, I want to avoid setting the minimum intensity value too high. If I was to set the minimum intensity value to 4000, for example, I would be excluding most of my fluorophore spots from being analyzed any further. Because of this, I would likely get very few pairs between the red and green channels of the movie. If I set the minimum intensity value too low, however, the only downside is that I would likely get a lot of pairs between the red and the green channels that were complete junk / noise. Note that the part of the histogram that is in green is the part of the intensity spectrum that will be considered for pairing with the red channel.