Hi everyone,
I am currently trying to validate two fluorescent proteins as a new FRET pair by Acceptor photobleaching. As the implemented bleaching function of my microscope software (ZEN 2009) doesnt work properly, I tried doing it manually by the following:
- Within my field of view I cropped into a little region of a proper cell, went into Live/Continuous mode and illuminated the region with 100% laser intensity for about 60-75sec
- As a negative control I measured 1-2 regions in other cells within the same field of view but with enough distance to the bleached region
All in all I bleached and analised about 15 regions this way and didnt get significant results. Now I wonder if my method is wrong and have many open questions that literature did not suffieciently answer:
-For how long do you usually bleach the acceptor respectively by how much %?
-Is my kind of negative control sufficient or do I have to transfect the Donor alone to get a real negative control?
-By how much % should the Donor fluorescence increase after acceptor bleaching in order to validate the FRET pair?
-Is Acceptor photobleaching alone even enough to validate a new FRET pair?
Thank you in advance :)
Check out this video on YouTube about FRET acceptor photobleaching. They use the Zen software so that you can relate the settings with your setup.
https://www.youtube.com/watch?v=CODwQO6TUB4
Check these other discussions on RG:
- https://www.researchgate.net/post/Minimum-FRET-efficiency-by-acceptor-photobleaching
- https://www.researchgate.net/post/How-to-avoid-donor-bleach-CFP-in-a-FRET-acceptor-photobleaching-experiment
- https://www.researchgate.net/post/How_can_I_calculate_FRET_efficiency_and_also_process_images_for_qualitative_analysis
- Article A quantitative protocol for dynamic measurements of protein ...
Good luck.
@Sathya Srinivasan
Dear Sebastian,
Illuminating a small region with full laser power for a minute is very long, but it obviously depends on how much acceptor bleaching you got with this long bleaching. If the acceptor is saturated (i.e. almost 100% of acceptors is in the excited state) at e.g. 50% laser intensity, there is not much point in increasing the laser intensity further. So the question really is how large fraction of your acceptor intensity was lost after your bleaching.
For a simple acceptor bleaching experiment you compare two intensities:
- donor intensity of the donor-acceptor sample before acceptor bleaching, Ida
- and the donor intensity in the same area after completely bleaching the acceptor, Id
Then E=1-Ida/Id
There are several assumptions that must be satisfied for this simple equation above to be applicable:
- no residual acceptor intensity and no acceptor recovery
- no bleaching of the donor
- no spectral crosstalk
In many cases these requirements are not met, and then you need to do more corrections. These can be found in this publication for example:
Article AccPbFRET: An ImageJ plugin for semi-automatic, fully correc...
Hope this helps,
Peter
A few things that might help. Try and keep your PMT gain low if you can since noise will mask small FRET changes.
Second, make sure your imaging conditions are not causing bleaching of the donor. Bleaching would also negate any increase in signal that you are looking for. Set up a time lapse acquisition exactly how you want to set up the bleach sequence, but leave out the bleach at first. I usually collect 2 or 3 pre-bleach images in both channels, and then at least several after. Make sure this sequence has little or no bleaching of the donor and modify settings if needed to prevent bleaching. Then go ahead and add the bleach step.
Third, your bleaching should reduce acceptor signal by 80%. Set your parameters to only what is needed to achieve that. I think a prolonged bleach my allow for diffusion and reduce the effectiveness.
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