I've been trying to measure different signals (calcium, mitochondrion) vs. the background. However, although the signal is quite clear to the eye, there is barely any difference if I look at the mean intensities on the spreadsheet. Could it be that too much signal is accounted for the background?
On another note, in the attached image you can see how I identify the secondary objects. This is in concordance with the general approach to just draw a ring around the nuclei and measure the signal. However, as the calcium is only presented on one site of the cell and not surrounding it, I'm also measuring a lot of background which weakens the value of the signal. I haven't found a more suitable algorithm to identify the 2nd objects... My questions is, is there a way to only select a part of the ring for measurement (e.g. highest 70% of the pixel) or can I somehow make the indentification algorithm better?
Here is the pipeline: autoPipeU2Otreated.cppipe
and an example image: https://drive.google.com/open?id=0B843VDZE-ww2bVBnY1QwLXRYY2c
Any help would be highly appreciated