Open ImageJ, then calibrate for analysis.

See http://imagej.net/ij/docs/examples/calibration/ for calibration setup steps and files.

Select Analyze > Calibrate, then select the following:

i. Rodbard (NIH Image)

ii. Unit: O.D.

iii. Open the calibration.txt file

iv. Uncheck ‘show plot’

v. Check ‘Global calibration’

Open an image and outline the regions of interest, including a background area for subtraction.

Select ‘multi measure’ and copy the results to an Excel sheet for analysis. Repeat for all images.

Take the ‘mean OD’ value for each region and subtract the background area ‘mean OD’ value. Values can be plotted directly or normalized to control groups.