I have tried what you recommended and I have used “Analyze -> Set Scale”, but I have noticed that the program gives me the same measures in pixel and in micron.
I have also tried the following getting the same results:
I opened a not-composite image (which shows measures in micron) and I used the Line Selection tool to draw a line. I measured the line with Roi Manager getting 52.944 um. Then I pasted the same line on a different image, a composite image (which shows measures in pixels) and I measured the line with Roi Manager again. The value of the measures I got was the same (i.e. 52.944) but pixels!
Then I have tried another method: using “Analyze -> Set Scale”, I have changed “Unit Length” from pixel to micron. I noticed that the report down below changes from “5.404 pixel/pixel” in “5.404pixel/micron”, but the value of the paths measured doesn’t change!
I have tried to do the aforementioned after reading it on this link https://imageJ.net/Spatial_Calibration, but I I do not have the parameters needed to calculate the line that I drew with the Line Selection tool: as written in the link above, they use a sort of “centimeter” to define how many microns the pixel measure line corresponds. I don’t have this “centimeter” and I don’t know another way to change the measure from pixel to micron. Do you know how can I get this “centimeter” on the image or if I need to use another parameter to convert the value?
According to the link above, I should change the “Know Distance” writing how many pixels long the line I draw is! But I don’t know this (dotted value in red)!
Given that the program measures the same value in the two different images (52.944 microns in the not-stitching image and 52.944 pixels in the stitching image), I wonder if the two measures are actually the same, and there is some kind of algorithm that is already doing the conversion, therefore I can consider as if 52.944 pixels were 52.944 microns when I analyze the stitching image (ultimately to measure dendrite length).
Let me know your thoughts on this, please