I am hoping someone can help me- I did a bunch of cytokine arrays on serum samples (40 cytokines dotted in duplicate plus controls for a total of 88 dots per blot- developed using HRP-chemiluminesence and captured with a digital imager). I have .tif's of both the dark and light images so that I can orient myself, but performing the analysis on just the unaltered dark image to retain vital pixel info. My question is regarding the viewing differences between CS6 and ImageJ. I had planned to use imageJ integrated density/histogram measurements by drawing a little circle around the dots on the unaltered file (actually drawing one circle and moving it from dot to dot so that everything is as consistent as possible) according to the layout of the blot. When I look at the original .tif in CS6 (this is where I made the overlay to orient myself) I can see more dots (positives) than I can see when I open the unaltered .tif in imageJ. That kind of stinks because I am looking for even the littlest bit of cytokine positivity that I can find. I am not trying to make quantitative measurements but rather qualitative "yes its there or no its not" measurements and comparing relative levels between samples after normalizing to total protein content. Why on earth does it seem like imageJ is losing information or subtracting background or something? The images are currently in 16bit grayscale (just the way the imager captured them). When I convert to jpeg or an 8bit .tif it shows up better in imageJ but stil not as clear as CS6- if I re-save as jpeg how much data am I losing? Alternatively is there a way in CS6 to retain a list of histogram data moving from point to point? Any insight would be greatly appreciated.
ImageJ loses pixel info???