Hi Colin,
best would be to place an object of know size beside the defect you want to measure.
I used to take an ASTM E1025 or E1742 plaque hole IQI, and put it on the object in the beam at the position where I located the defect.
Then I used the line profile function in the YXLON software Image 3500 and calibrated the pixel size by the IQI dimension at the position of the defect:
The E1742 penny is 2" long (50.8mm or 50800µm for higher precision)
In this example I simulate the defect with the 4T hole of the IQI. For the hole size I read in ASTM E1742:
Minimum diameter for 4T hole = 0.040 in. which is 1.016mm (or 1016µm).
I open the line profile function in the YXLON software, make the Subpixel function active and set as start point the left side of the hole (green arrow):
Now I move the mouse pointer to the right side of the hole and see the dimension 1039.1µm (which is quite near to the nominative 1016µm)
I know that measurement in subpixel range is very sensitive on noise ... (but offers the highest resolution for measurement in the image)