Figure 2. Example of a, IJM language macro used to create a color threshold for PLA reactions in a fluorescent image.

Because the image used was only the PLA fluorescence instead of the merged image, min and max settings for the hue and saturation could be inclusive (Lines 14, 15 for Hue; lines 17, 18 for saturation). Minimum brightness was adjusted to eliminate background while including signal (Line 20). Macro creates a mask and counts particles remaining after the threshold is applied. Macro may be used to count particles from multiple image files at once by use of the batch function built into ImageJ.