We have observed a fluorescence recovery some minutes after a fluorescnce decrease by a light exposure of GFP and its derivatives some minutes some minutes after a fluorescnce decrease by a light exposure. Clearly, this is not a simple photobleaching of chemical fluorescent dye. How does it happen? I think that this is a kind of photochromism, however, I cannot find any literatures which precisely explain this fluorescence recovery of GFP.
Was this experiment done under conditions in which new GFP synthesis was occurring?
Yes. Usually, the cells (COS7 or HeLa cells) are observed by time-lapse microscopy 48 hour after the transfection with the plasmids encoding GFP-fusion proteins (or YFP/CFP-fusion proteins) . GFP proteins begin to express around 18 hour after transfection and reach the plateau around 36 - 48 hour after transfection. So, I think that synthesis and degradation of GFP are in a condition of balance when I observe the cell 48 hour after transfection.
In that case, the fluorescence recovery could be due to resynthesis.
Thank you for your response. However, experimental details deny the explanation. My experiment is a ratiometric FRET imaging, so a FRET sensor contains both YFP and CFP. Just after the selection of a target cell (This means a 10 - 20 seconds continuous exposure to an excitation light), the fluorescence from CFP and YFP in the sensor in this cell will be reduced by 10% in comparison to the basal level. (In this case, I measure the total fluorescent intensity in this cell). Then, if I start the 1 minute-interval timelapse in an appropriate condition immediately, the total fluorescence from CFP and YFP in the sensor in this cell will recover to the a specific constant value during suceeding 5 - 10 min and maintain this constant value with a slight fluctuation (at least two hour after the beginning of the timelapse). In other words, the total fluorescence from CFP and YFP in this cell does not continue to increase in this situation. Thus, I think that the fluorescence recovery cannot be explained by resynthesis of CFP and YFP, at least in part. In practice, after the selection of a target cell, I usually wait for 5 min. In this case, the total fluorescence from CFP and YFP will keep a constant level from the beginning of a timelapse experiment.
Hi Takeshi. You're probably right that it's not simple photobleaching. GFP and the other FP's all seem to undergo a photoconversion process during excitation during which they typically shift to a dimmer red-shifted shorter lifetime state. But even in the ground state they are at an equilibrium between these two states, which is the basis behind why these FP's show biexponential lifetimes. It's possible your sample was simply returning to this normal equilibrium. I'm attaching a link that discusses these states, conversions, lifetimes, and the equilibrium. http://www.sciencedirect.com/science/article/pii/S0006349505732562
Hi Ryan. Thank you for your excellent comment. I am now reading the Biophysical J's paper that you mentioned. The idea of a photoconversion and a return to a normal equilibrium naturally fits with my feeling based on my experience.
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