Fig. 3Thresholding parameters for counting spots in the FindFoci ImageJ plugin. With these settings (a) the
“Background param” slider is adjusted until all spots are identified in the image. The FISHQuant and Imaris
Spots applications use intensity thresholding (b), which is used to provide an initial separation between
background and high intensity spots (arrow), which are then refined using additional parameters
Super Resolution smFISH in theDrosophilaNMJ 171