Hi all,

I'm staining live neurons with Fluo-4 AM esters (Thermofisher cat# F14201) and seem to be having some issues when capturing images. With video recording with continuous excitation, there seems to be gradual decay in signal. However, with images taken every few seconds as a timelapse, there is instead a gradual increase in fluorescence with the same cells. Neither of these are really what I expect to see, since even if the cells are doing nothing the signal should be relatively constant.

Has anyone worked with Fluo-4 AM and know whether the dye exhibits signal decay with photobleaching, or been able to nail down a way to reduce these fluctuations? I've worked through the troubleshooting tips on Thermofisher's site.

Thank you!