A problem that arises occasionally is a slow shift of our well during our experiments. These experiments routinely last 2 to 3 hours. During this time,
The entire well will shift 5 - 10 uM diagonally to the top right or bottom left. This means the cell will be half inside and half outside of the ROI by the end of the experiment.
The FURA2 will gradually (but not completely) bleach. Thus, the intensity will be less at the end then at the beginning.
We need to use the ROI to obtain additional measures. For example we image GFP and mCherry at the end of the experiment.
This gives us a picture of each individual cell like below;
I wanted to message here to get an idea on this group would approach this complex issue.