Rationale for establishing thresholds to differentiate between background and true-positive fluorescence signals. Panel A: Beginning with raw data, the histogram method transforms the (X,Y) data into polar coordinates, (r,θ) (Panel B). Panel C: From the polar coordinates, the method generates a histogram of the magnitude, finds the first minimum (magnitude threshold), and removes the negative samples. Then the method generates a histogram of the angular components and determines the first and last minima as the two angular thresholds. Panel D: Finally, the thresholds are transformed back to Cartesian coordinates, where the angular thresholds become lines and the magnitude threshold becomes a quarter circle.