Recently, my lab has run into an issue with calibrating B/W photomicrographs of picrosirius red (PSR) stained sections in ImageJ. We photograph sections of stained tissue with polarized light and use optical density (pixel value -> calibrated OD) to measure the amount of light in the tissue as a way to measure collagen content. We are averaging a number (9) of areas in a picture together to get an average value (as opposed to measuring the whole picture).
Before, we would use the Rodbard function inside ImageJ's calibrate window with numbers that our lab had generated for our microscope. However, when we use this now, the curve is distorted and (appears) to be returning the pixel value instead of the OD value it should be (see attached document for screenshots of this). It also looks like the equation may have changed.
As for a solution, we found that using the Uncalibrated OD function produces the correctly shaped curve as well as reproducible results (we reran some old photographs/data and slides and came out with comparable numbers).
Is this solution a good one/can I use the Uncalibrted OD in this way? Has anyone else experienced this error? Your insight is greatly appreciated. Thank you
See attached document for screen shots of my issue (before and after the error occurred) as well as my solution with supporting references for using the Uncalibrated OD curve.