Muscle Cross-sectional Area Measurement with ImageJ Software (Dr. RATCHAKRIT)
Purpose: This approach is written to assist the researcher manually determine the cross-sectional area
(CSA) of muscle fibers with an open-source software (ImageJ*). The representative images were taken
from Nikon Eclipse 600 at 200× magnification. If you use different microscope and magnification, you
need to change the values in item no.2 (Distance in Pixels and Known Distance).
Distance in Pixels = the length of scale bar that is measured in pixels by drawing a straight line using Straight tool
to determine the actual length of scale bar and then measure it with Image J software (Analyze/Measure); Known
Distance = known size of scale bar (µm) at a specific magnification.
*Image J can be downloaded at http://imagej.nih.gov/ij/download.html (version 1.44 is recommended).
Image J software developer: Wayne Rasband ([email protected]), National Institute of Mental Health, Bethesda, Maryland, USA.
1. Open ImageJ software and click File tab, then open an image that want to measure CSA.
2. Select Analyze tab/Set Scale, then type 204 (Distance in Pixels), 100 (Known Distance), and type
Micron for unit of length. Check mark on Global to apply these setting to all measurements.
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3. Close the Set Scale Dialog Box and select Freehand selections tool on the toolbar, then draw the
CSA of the muscle fiber and the yellow line will appear.
4. Select Analyze/Measure, then the Results Dialog Box will pop-up.
5. After the result is obtained, select Process/Noise/Add Noise, then the gray noise will appear on
the measured fiber to avoid repeat measurement in the same fiber again.
Note: If H&E-stained section is used, select Process/Find Edges function instead of Process/Noise
H&E-stained image
6. After finish the measurement, select File/Save As in Results Dialog Box and then set the file name
that want to save. The result file (.csv) can be opened later with Microsoft Excel.
Dr. Ratchakrit Srikuea
Department of Physiology, Faculty of Science
Mahidol University, Thailand