I have some confocal z-stacks of cells expressing a fluorescent  
protein and I've encountered something I haven't had to deal with  
before. I'm wondering what is the best way to subtract the background  
signal from a z-stack like this. My problem is that the background is  
different for each z-step, peaking a the z-height corresponding to  
the bottom of the cells that are in contact with the substrate -  
supposedly since this is the height where you have the most  
reflection of the excitation light from the substrate itself  
backwards to the detector.

Which is better? Should I subtract the maximum background level of  
the substrate slice from all slices or subtract the background of  
each individual slice from themselves only? Or I could subtract the  
average background over all slices from each slice instead of the  
maximum value at the substrate slice. Alternatively, should I create  
the z-projection and subtract the background of the z-projection image?

Thanks for anyone who can clear this up for me.


John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and Biomedical Engineering
Centre For Studies in Molecular Imaging