I have some problems to convert green mEOS2 to red. I have been using the confocal laser at 405 without good results. I got some red fluorescence which is photobleached after some minutes (not continuous excitation... the acquisition was every 10 sec) and it is supposed to be more stable than the green version of the protein)
could any body give me some advices?
Though more stable, the red fluorescent version still will photobleach rather quickly. That is the problem in general with fluorescent proteins. I do single molecule imaging using mEos2 and generally the fluorescence only lasts a few frames, so your photobleaching time is what is expected. I attached a publication that goes over the photobleaching properties compared to other fluorescent proteins.
Article Charaterization and development of photoactivatable fluoresc...
Thank you very much Kelsey, I will take a look.
By the way... Which power of laser do you use?
I use a fairly high laser power; we use 568nm as our excitation wavelength at a power of 25mW and an exposure time of 20-50 ms. If your exposure time is much longer than that, you can use much lower powers. So if your exposure time is closer to 500ms, you could try 5mW or maybe even 1mW, depending on the expected mobility of your protein. Hope that helps, good luck!
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