I saw you are so helpful with this so I thought you might provide some insight to my problem similar to Abdelhady.
Attached below is my aorta in H&E staining, 40x. I captured the image using Olympus microscope in JPEG, not knowingly that TIFF is actually the best format for ImageJ, hence the unit given is in pixels instead of micrometer.
I want to measure the intima thickness, shown with black line, it starts at the first upper layer to bottom lowest layer.
Tried to calibrate by following exactly like Getting Started page instructed, but I just CAN’T change any number or unit in Set Scale dialog box, nothing happened.