Re:Ion intensity for ROI

[David Pirman]

Ramin,

So for quantitation, it can be a bit tricky, but you are correct. You should definitely think about ng/area and then even volume (since you know your tissue thickness). I generate standard curves by spotting calibration standards beneath my tissue (in a fixed circular area) average the intensity within the spot and then calculate the volume of tissue. Then I would quantify with same exact area size somewhere else on the tissue, but I haven’t had much luck in this without an internal standard. So in my case, my calibration standards were in a nice confined area with a uniformly applied internal standard. If you are trying to generate quantitative data from a compounds with unknown area density information, I’d think it would be quite difficult since the calibration curve is heavily dependent on the calculation of area on the x-axis. I just had a paper accepted in Analytical Chemistry using this technique for quantitation.
For calculating the area accurately in DESI, I would suggest drawing an ROI and overlaying the image onto an optical image of your tissue and basically doing a manual measurement of the area you are interested in. Also, placing some distance registration marks on the MALDI target. I typically use glass slides, and will simply draw marks of interest on the backside so when I take an optical image I can make measurements and co-register the MS image.

Dave