Whilst calibrating my Motic Microsope's camera, I am able to calibrate the 10x object lens against the stage micrometer accurately. However when I calibrate (using the Motic 2.0 Plus software) the 40x object lens, it always sets the ratio incorrectly.
For example, in the calibration panel, I load a photo of the scale that I took and then I draw the circle so that it measures the pixels both in height and width and then I tell it that the circle is 10 µm. But it doesn't seem to get the ratio correct. When I did this for the 10x lens it worked perfectly.
So I took a photo of the scale with the 40x object lens and then went to calibrate that.
Here you can see how the calibration is performed: you position a circle over the photograph and tell it the size in µm (it's rather large, sorry):
Here you can see the measuring line out of whack. It gives me 8 µm for a length that should only be 4 µm.
Am I missing something?