While visualizing the green fluorescence intensity of oocytes stained with DCF staining, we notice that the intensity continuously and rapidly increase once you start the excitation. Any idea why is that happening?
It seem to be auto-oxidation. Anti-fade agents could help to decrease observed effect.
If it possible try to perform optimization of excitation light (attenuation of intensity, field, filter set, diaphragm). As option use usual illumination (non-fluorescent) to identify correct position and cell and switch to fluorescent illumination only for short period to get images.
Have a nice day!
Daniil
It is possible that you generate ROS just by exciting the fluorophore. Furthermore is the reaction of a ROS sensor not a static readout and does not necessarily reach a steady state. We used CellRox in cells and compared the increase kinetics between treated and untreated.
Autoactivation is a well known problem with all DCF based dyes, which is why reviewers increasingly don't like them. Although it's a lot more work, expressed sensors such as roGFP and roGFP-orp1 do not exhibit this problem. Avoid hyper-based expressed sensors because they are very pH sensitive.
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