Hi, I'm new to ImageJ software and I'm using it to quantify cell area (~50-80 cells per image). The area should be roughly 300-500 um^2 but when I quantify, the area comes out to 12-16 um^2. I went over my method with the postdoc that trained me. We're doing it the same way and getting different results. Here is my method:
1. Use straight line tool on scale bar, Set Scale->input known distance
2. Set Measurements, select "Area"
3. Freehand tool, manually trace cell boundaries
4. Measure
Is there something wrong with my settings that may be causing this?
Hi Brent S. I don't see any error in your method. The only error possible here is the estimation of the known distance of the scale bar. This may be due to the scale bar in the microscope not calibrated or incorrectly calibrated. Either recheck that and repeat or do the following as an alternative approach:
1. check the sensor size (SS) of the microscope camera (usually 4-10 um). This will be available on the online manual of the cam.
2. Calculate this Value=SS/M. where M is the objective magnification (10x, 20x etc)
3. After opening the image on ImageJ, go to set scale, and manually set the scale bar. Distance in pixels=1, known distance=Value.
4. Follow your steps 2 through 4 as usual.
Hope that helps.
BG
I agree that it sounds that your scale is incorrect. The suggestions above are good; if your microscope doesn‘t have a sensor size, you can photomicrograph a standard measuring bar and manually calculating your pixel/micrometer ratio.
- Badges
- Science topic
- Similar topics
- Computer Science
- Images