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Dear Kees,

Welcome to murky world of FRET imaging! For those researchers who have taken the time to compare different FRET microscopy methods (such as myself) on the same biological system, I would suspect they would say this is not an uncommon result. I too was never able to get the same FRET efficiency values when I performed acceptor photobleaching and sensitized emission imaging (in my case intermolecular FRET as measured by CFP and YFP labeled versions of the same GPI protein - we were looking for oligomers of the protein in the cell membrane). There have been a number of papers reporting/debating the conversion of YFP into a "CFP-like" emitter after photobleaching. For example, see here:

Valentin, G., et al., Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments. Nature Methods, 2005. 2(11): p. 801-801.

Thaler, C., et al., Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements. Nature Methods, 2006. 3(7): p. 491-491.

Valentin, G., et al., Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements - response. Nature Methods, 2006. 3(7): p. 492-493.

Verrier, S.E. and H.D. Soling, Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements. Nature Methods, 2006. 3(7): p. 491-492.

Seitz, A., et al., Quantifying the influence of yellow fluorescent protein photoconversion on acceptor photobleaching-based fluorescence resonance energy transfer measurements. Journal of Biomedical Optics, 2012. 17(1).

I am currently participating in the annual Quantitative Imaging course at the Cold Spring Harbor Laboratory in New York. Two days ago we had a nice lecture by George Patterson on the history and application of fluorescent proteins in molecular biology. I asked him what the latest was on this topic and he said that not everyone observes the YFP to CFP-like conversion. The process is not understood well and he thinks it may depend in part on the molecular environment of the probe in each case. He also mentioned that researchers he had met who in the past had not observed the process at some time later said that better detection methods showed the process was occurring at some level in their studied systems. I think the most interesting fact I gained from this lecture was that many of the natural and engineered fluorescent proteins undergo some kind of photoconversion or photoactivation process (he calls them optical highlighters). He emphasized that one should pay attention to the lineage of the fluorescent protein they're using since some of the mutations and even the species of origin of the fluorescent protein lead to stronger photoconversion/photoactivation properties, ie: some YFPs are better than others when it comes to avoiding this effect. It is for this reason and my past personal experience that I am wary of acceptor photobleaching FRET methods now. 

The difficulties associated with measuring sensitized emission FRET don't make the situation any better, but I would be more inclined to believe this type of experimental result if the control experiments are done well and the instrumentation performance is checked (image registration, bleed-through correction, etc.). I think the experts out there on FRET microscopy would say that the better approach is to turn to time-resolved FRET measurements like FLIM, but of course these methods are more costly and complex sometimes. Lastly, I would point out there is another lesser known FRET imaging method that involves imaging polarization changes in one fluorescent protein rather than detecting intensity changes between two fluorescent proteins. This method is called "homogenous FRET" and has been shown to be one of the more sensitive methods to detect the presence of FRET, but it does not yield an efficiency value. I have used it before, and it agreed with my sensitized emission FRET results. The method is reviewed in this paper by Dave Piston:

Piston, D.W. and M.A. Rizzo, FRET by fluorescence polarization microscopy, in Fluorescent proteins, second edition. 2008, Elsevier Academic Press Inc: San Diego. p. 415-430.

Cheers,

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2013-04-09, at 5:11 AM, Straatman, Kees R. (Dr.) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> *****
> 
> Dear list members,
> 
> Last week we did some FRET experiments using CFP-YFP on a Leica SP5 system. The idea is that conformational change of the tagged protein will result in FRET, so ratio CFP:YFP is 1:1. 
> 
> We did both Acceptor Photo-Bleaching and  Sensitized Emission experiments on the same samples and we get results I cannot explain. 
> 
> For CFP we used a 405 nm laser line, for YFP a 514 nm laser line.
> 
> With  FRET Acceptor Photo-Bleaching following the Leica wizard we get an increase of CFP signal of around 22% but the YFP control shows a conversion to CFP-like signal of around 16% so this leaves us with a FRET efficiency of ~ 6%. Not really impressive. 
> The bleaching has no measurable effect on the CFP control. I also tested if there are changes over time in the CFP signal after Acceptor Photo-Bleaching (like a FRAP experiment) but found no changes.
> 
> However, with FRET Sensitized Emission following the Leica wizard or doing manual imaging of 7 different images we get a FRET efficiency between 30-40%. We tried to use for the different controls cells with the same intensity in CFP or YFP. We also compared cells with bright and weak signal but found no difference in results. 
> 
> We repeated both experiments to check if we had an influence of timing on the conformational change of the tagged protein but got the same results.
> 
> Obviously, the user wants now to know what is the correct result and why? I have thought hard about it but have no answer. I seem to miss something somewhere. I have to add that we don't often do FRET and in most cases we only do Acceptor Photo-Bleaching.
> 
> Does the conversion of YFP to a CFP-like signal not take place in the presence of CFP? 
> 
> For Sensitized Emission different cells are used in the different samples to make the calculation but the results are consistently much higher, so cannot explain the difference.
> 
> Does somebody else have found this type of differences between FRET Acceptor Photo-Bleaching  and FRET Sensitized Emission using CFP-YFP? And can somebody explain the results we obtained?
> 
> Many thanks and best wishes
> 
> Kees
> 
> 
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Centre for Core Biotechnology Services
> University of Leicester
> http://www.le.ac.uk/biochem/microscopy/home.html