Hello!
I was recording photobleaching curves for Alexa Fluor 647 fluorophores bound to an Al2O3 coated surface in a buffer solution (containing no fluorophores), and found that average fluorescence in an area is increasing or staying constant for first >10 seconds in the beginning of bleaching, and starts going down only later on. See the attached curves, with an arrow showing this bulge in every curve. Can anyone suggest why does it occur? Can that be some kind of dequenching? Thank you for your advice.
I'm using a 640 nm laser for excitation and a microscope with EMCCD camera for imaging.
Hi Ivan Unksov , Isn’t this observation because you are increasing the laser power. As a result more molecules will be retain in the excited state to undergo emission which results higher intensity due to increased laser power. also it is important to pay attention to the slopes of your curves. The blue curve has a larger slope than the black curve which clearly indicated the photobleaching ability of the dye is more obvious under higher laser power.
Hi Chathura Abeywickrama , thank you for your answer, however I meant not the rising intensity with excitation power but the bulge in each of the curves in first 50 s of bleaching, the time region marked with dashed line in my figure. I don't understand why in the beginning of bleaching process the intensity in every curve firstly goes up (or remains constant in the 30% curve), and only after that it starts decreasing. Sorry if my phrasing was not accurate, I rephrased the question now.
Hi Ivan Unksov,
If you have the possibility to measure the fluorescence lifetime during the bleaching, you may have the chance to see an increase of the fluorescence lifetime and yield during the first 30% of your bleaching. It has been predicted that in assemblies of fluorescent molecules where the excitation can move, it can move toward non fluorescent traps. If the traps are more fragile than the fluorescent molecule of the antenna, the bleaching will purge the assembly from its traps and the fluorescence yield will increase. You can have an indication of the presence of diffusion of the excitation among the fluorescent molecules by measuring the anisotropy of the fluorescence.
Hello Robert Bernard Pansu , thank you for your answer! Do you mean the acceptor traps described by D. L. Huber in Phys. Rev. B 20, 2307 ? Do you think some molecules of the fluorophores population can act as the traps, like in homo-FRET?
Homo- FREt can indeed transfer the excitation toward dimers of Alexa Fluor 647 that are non fluorescent. I am ready to bet that Alexa Fluor 647 dimers are non fluorescent because of a photoinduced electron tranfer between the two members of the pair. Then it looks resonable that during the short time when the fluorophore are cation and anion they can decompose. But this is not facts, this is imagination. But you can check that Alexa Fluor 647 dimers are non fluorescent. You can check through a Forster cycle that photoelectron tranfer is possible. (If you can find the redox potential. You can checkk that the number of traps increases with the density of labelling. Then your result could be a good indication that the dimers bleach more quickly than the monomers.
please have a look to
(2010) M. Martini, S. Roux, M. Montagna, R. Pansu, C. Julien, O. Tillement and P. Perriat. How gold inclusions increase the rate of fluorescein energy homotransfer in silica beads. Chemical Physics Letters. 490, 1-3, 72-75. http://dx.doi.org/10.1016/J.Cplett.2010.03.027
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