I am new to the image J forum.
I am also measuring flourescence intensity, so would like to check my protocol and ask some thinks I am not sure about.
I need to compare caspase-3 staining between two groups of cells (control and patient cell lines). Rather then comparing intensities of the individual cells, I need average intesity of all the stained cell nuclei across the image.
Here is what I am doing:
- I did z-stack imaging, so first I creat a z-projection using max intensity method.
- Then I split channels to get the channel that shows just caspase staining
- Duplicate the image
- Adjust threshold manually on duplicated image (I use fill holes and watershed option if necessary)
- I enabled limit to threshold in set measurements and redirected measurements to the first image
- Then I do measurements with analyse particles (I have chosen both mean gray value and integrated density to measure, but I think integrated density would be a preferable choice). I have checked summarize box to get mean integrated density of all the measured cell nuclei in the image.
Images are calibrated.
- I was using manual threshold. However I am a bit confused now. It seems that both manual and auto threshold could produce biased measurements. If I use manual threshold with always definied range would that be ok? Or is there a certain auto threshold method that wouldn’t produce biased results on my images?
Just to reflect on the problem that was mentioned in the comments: I was using the same channel for threshold and measurements, but I can see I will have to use DAPI to define threshold, and do the measurements on the caspase-3.
- Should I measure the intensity of the background too as they did it here:
Here is example image:
I would be greatful if somebody could clear this things for me