So I’ve read almost all forums I can find on here about measuring fluorescence intensity to try and find the best option…I am trying to compare intensity in different conditions and then compare the intensity of multiple cells within a condition
I’m just wondering if I’m doing the same thing twice (subtracting too much background maybe), here’s my suggested workflow:
- SUM project 40x zstack
- Subtract the background with a rolling ball radius/sliding paraboloid
- Select ROI of cell and take measurement (Mean gray value, Area, integrated density)
- Select an ROI of background near the cell according to this method to get the corrected total cell fluorescence (CTCF).
I’m just wondering if step 2 and 4 are doing the same thing, and whether I should do both or just choose one.