Hello everyone, I am measuring wheat root anatomical traits (whole root diameter, stele diameter, etc.) using ImageJ. Initially, I used a compound microscope at 4x magnification and calibrated the scale using a physical stage micrometer (ladder). Due to a change in equipment availability, I switched to a dissecting microscope for a few remaining samples. This microscope automatically burns a 1 mm scale bar into the image. I used this built-in scale bar to set the scale in ImageJ, also at 4x magnification. However, the measurements from the dissecting microscope are consistently and noticeably higher than those from the compound microscope. I followed the standard “Set Scale” procedure in ImageJ for both sets of images. What could be causing this measurement discrepancy? Are images taken from two completely different microscope types fundamentally comparable for this type of anatomical data, even if both are at 4x? Any insights would be greatly appreciated!
Hi Ishrat,
Both microscopes should measure length accurately. My guess is that the 1 mm burned into the image by the dissecting scope is off. Can you image a stage micrometer (or any good mm ruler) on that scope to check?
As ever,
Tobias Baskin
In addition to what Tobias is suggesting, if you trust the optics to be effectively at 4X magnification (i.e. the microscope looks like original and hasn’t been modified) and you can see the camera. Another thing you can do is figure out the physical pixel size of your camera (by searching the camera data sheet), and divide this size by 4 (the magnification). This gives you your pixel size in the image, and while not as precise as measuring a “stage micrometer”, it should give you a good estimate.
For example, if the camera data sheet reports a Pixel Size of 2.74um:
And your magnification is 4X, then in your image, the pixel size should be 2.74/4=0.685um. As a result, a 1-mm scale bar should span 1000/0.685=1460 pixels. I picked a random camera so don’t quote me on the exact numbers!
Take care,
Omni
UPDATE—solved. The burned-in scale bar was wrong.
Thanks everyone.
I photographed my stage micrometer on the dissecting scope at 4x and calibrated from it in ImageJ. The 100 µm divisions came out at 185.2 px, so 0.540 µm/px. The burned-in “1 mm” bar is 466 px, which works out to 2.146 µm/px. That’s 3.97 times too large, which is exactly why everything measured about 4x bigger than on the compound scope. I shot the micrometer at 4x and at 10x. The burned-in bar is 466 px in both. A real 1 mm bar would have to be 2.5 times longer at 10x. It never changes, so it was never measuring the specimen.
Never trust a burned-in scale bar you didn’t verify yourself, and photograph the micrometer at every magnification you actually use.
You were right Tobias
Thank you for letting me know.
Yes, I have had a long distrust of those ‘burned in’ things. I always like to measure myself. Also those burned in bars sometimes get in the way of something nice in your image!
Take care,
Tobias