FRET Acceptor Photo-Bleaching vs Sensitized Emission

[Straatman, Kees (Dr.)]

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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

[John Oreopoulos]

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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:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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

[mmodel]

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My understanding that in acceptor photobleaching you can only know that you have destroyed fluorescence of the acceptor, and it is not the same as destroying its absorption band (which is responsible for FRET). Absorption can be more resistant to photobleaching than fluorescence.

Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Straatman, Kees R. (Dr.) [[hidden email]]
Sent: Tuesday, April 09, 2013 5:11 AM
To: [hidden email]
Subject: FRET Acceptor Photo-Bleaching vs  Sensitized Emission

*****
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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

[Cedric Espenel]

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Dear Kees,

You probably found your answer in the previous messages, I will just had that you might want to try using FLIM to measure your FRET efficiency. Although the analysis is not always straight forward in cells it will avoid a lot of your actual problems.

Best

Cedric

---------------------------------------------------------

Cedric Espenel, Ph.D.
Post-Doctoral Associate
Dept. of Cell and Developmental Biology
Weill Medical College of Cornell University
1300 York Avenue, Room A-201
New York, NY    10021

212-746-6079 (lab tel)
212-746-8175 (fax)




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: mardi 9 avril 2013 05:11
To: [hidden email]
Subject: FRET Acceptor Photo-Bleaching vs Sensitized Emission

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

[yuansheng sun]

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Dear Kees,

I am assuming you are doing live-cell measurements.  If you have the Argon
laser, I would use the 458 nm laser line for CFP instead of the 405 nm
diode laser, since it helps reducing phototoxicity and avoiding the
photo-conversion issue (if it is there).  If you use a strong power of the
405 nm laser, there is a possibility to get cellular NADH, which has a
small absorption rate at the 405 nm and emits in the CFP channel.

For your apFRET expeiments, you did controls with donor-alone and
acceptor-alone cells, which are good.  It is also worth doing a control
with the cells alone (untransfected).  Take the donor-channel images before
and after the bleaching, and make sure to apply the same bleaching
condition as used for your CFP-YFP samples.  Then you know if the
sample gives autoflurescence before and after the bleaching.  I assume you
carefully checked the pre- and post bleach images and made sure there were
no cellular movements and focus drifts.  These are common problems for
apFRET applied to live cells.

For the seFRET experiments, the FRET efficiency (E) is calculated based on
the quenched donor and FRET signals. Between the two, a correction
factor needs to be applied, considering different quantum yields, detector
sensitivities and optical light-pass efficiencies for the donor and the
acceptor.  I believe that the Leica seFRET module uses the Gordon et al
BJ1998 (2702-2713) algorithm, in which it is called the G factor.  You can
scale up or down all the Es with the G factor.  There are different ways of
calibrating this factor (Chen et al BJ 2006 L39; Sun et al JBO 2010
15-2).  If you want to compare the seFRET E with the Es obtained with other
FRET techniques (e.g. apFRET and FLIM-FRET), it is important to calibrate
the G factor in an experimental way.  However, if you look at relative Es
at different conditions or samples within a same assay, you can
theoretically estimate the G factor and use the same one for all your data
analysis.

Sheng
Yuansheng Sun, Ph.D.
W.M. Keck Center for Cellular Imaging
http://www.kcci.virginia.edu/workshop/workshop2014/index.php





On Tue, Apr 9, 2013 at 5:11 AM, Straatman, Kees R. (Dr.) <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>

[Straatman, Kees (Dr.)]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Thank you all very much for your replies.

I am well aware of many of the problems associated with FRET and in general if we do abFRET to say yes or no to an interaction. But because this experiment gave us a result that was very much on the border I decided to have a look at seFRET and this gave substantial different result. Because we normally use abFRET I was wondering if we might have made the wrong conclusions in the past.

This is indeed on live cells. Unfortunately, we don't have FLIM at hand, but I think I will give spectral imaging a go to see if there is really a FRET peak.

Will see if the 458 nm laser line gives a different result will check the gain settings so we stay in the linear area. The user will also make a control  cell line that will lack the protein that causes the conformational change in the CFP-YFP- tagged protein and therefore is expected to give no FRET, but I should underline 'expected' in this case...

Best wishes

Kees

   

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of yuansheng sun
Sent: 09 April 2013 20:02
To: [hidden email]
Subject: Re: FRET Acceptor Photo-Bleaching vs Sensitized Emission

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Kees,

I am assuming you are doing live-cell measurements.  If you have the Argon laser, I would use the 458 nm laser line for CFP instead of the 405 nm diode laser, since it helps reducing phototoxicity and avoiding the photo-conversion issue (if it is there).  If you use a strong power of the
405 nm laser, there is a possibility to get cellular NADH, which has a small absorption rate at the 405 nm and emits in the CFP channel.

For your apFRET expeiments, you did controls with donor-alone and acceptor-alone cells, which are good.  It is also worth doing a control with the cells alone (untransfected).  Take the donor-channel images before and after the bleaching, and make sure to apply the same bleaching condition as used for your CFP-YFP samples.  Then you know if the sample gives autoflurescence before and after the bleaching.  I assume you carefully checked the pre- and post bleach images and made sure there were no cellular movements and focus drifts.  These are common problems for apFRET applied to live cells.

For the seFRET experiments, the FRET efficiency (E) is calculated based on the quenched donor and FRET signals. Between the two, a correction factor needs to be applied, considering different quantum yields, detector sensitivities and optical light-pass efficiencies for the donor and the acceptor.  I believe that the Leica seFRET module uses the Gordon et al
BJ1998 (2702-2713) algorithm, in which it is called the G factor.  You can scale up or down all the Es with the G factor.  There are different ways of calibrating this factor (Chen et al BJ 2006 L39; Sun et al JBO 2010 15-2).  If you want to compare the seFRET E with the Es obtained with other FRET techniques (e.g. apFRET and FLIM-FRET), it is important to calibrate the G factor in an experimental way.  However, if you look at relative Es at different conditions or samples within a same assay, you can theoretically estimate the G factor and use the same one for all your data analysis.

Sheng
Yuansheng Sun, Ph.D.
W.M. Keck Center for Cellular Imaging
http://www.kcci.virginia.edu/workshop/workshop2014/index.php





On Tue, Apr 9, 2013 at 5:11 AM, Straatman, Kees R. (Dr.) < [hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>

[Tim Feinstein-2]

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*****

Hi Kees,

Apologies, clearly I responded to the discussion rather than to your original post.  I should have noted that you are using an intramolecular biosensor.  Of course that makes things simpler and your puzzle more of a puzzle.  The (theoretical) negative control cells will (theoretically) be a great help.  As you no doubt know by now not all biosensors that should FRET do FRET, and making a good one can take trial and error.  

Spectral imaging will help only a little if you excite with 458 nm.  Although the Mol Probes Spectra Viewer says that YFP excites with 458 (2% efficiency) and 442 nm (1%), in our hands cross talk is quite significant with a 458 nm laser and negligibly close to zero at 442.  You should see a YFP peak in this case regardless of whether transfer is happening.  

Question: does anyone know whether the 405 nm line is more likely to cause photoconversion?  I have little experience with FRET at 405 but I'd be cautious about using high-energy, low-efficiency excitation for this experiment.  

cheers,


TF

Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Apr 11, 2013, at 5:45 AM, Straatman, Kees R. (Dr.) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thank you all very much for your replies.
>
> I am well aware of many of the problems associated with FRET and in general if we do abFRET to say yes or no to an interaction. But because this experiment gave us a result that was very much on the border I decided to have a look at seFRET and this gave substantial different result. Because we normally use abFRET I was wondering if we might have made the wrong conclusions in the past.
>
> This is indeed on live cells. Unfortunately, we don't have FLIM at hand, but I think I will give spectral imaging a go to see if there is really a FRET peak.
>
> Will see if the 458 nm laser line gives a different result will check the gain settings so we stay in the linear area. The user will also make a control  cell line that will lack the protein that causes the conformational change in the CFP-YFP- tagged protein and therefore is expected to give no FRET, but I should underline 'expected' in this case...
>
> Best wishes
>
> Kees
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of yuansheng sun
> Sent: 09 April 2013 20:02
> To: [hidden email]
> Subject: Re: FRET Acceptor Photo-Bleaching vs Sensitized Emission
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Kees,
>
> I am assuming you are doing live-cell measurements.  If you have the Argon laser, I would use the 458 nm laser line for CFP instead of the 405 nm diode laser, since it helps reducing phototoxicity and avoiding the photo-conversion issue (if it is there).  If you use a strong power of the
> 405 nm laser, there is a possibility to get cellular NADH, which has a small absorption rate at the 405 nm and emits in the CFP channel.
>
> For your apFRET expeiments, you did controls with donor-alone and acceptor-alone cells, which are good.  It is also worth doing a control with the cells alone (untransfected).  Take the donor-channel images before and after the bleaching, and make sure to apply the same bleaching condition as used for your CFP-YFP samples.  Then you know if the sample gives autoflurescence before and after the bleaching.  I assume you carefully checked the pre- and post bleach images and made sure there were no cellular movements and focus drifts.  These are common problems for apFRET applied to live cells.
>
> For the seFRET experiments, the FRET efficiency (E) is calculated based on the quenched donor and FRET signals. Between the two, a correction factor needs to be applied, considering different quantum yields, detector sensitivities and optical light-pass efficiencies for the donor and the acceptor.  I believe that the Leica seFRET module uses the Gordon et al
> BJ1998 (2702-2713) algorithm, in which it is called the G factor.  You can scale up or down all the Es with the G factor.  There are different ways of calibrating this factor (Chen et al BJ 2006 L39; Sun et al JBO 2010 15-2).  If you want to compare the seFRET E with the Es obtained with other FRET techniques (e.g. apFRET and FLIM-FRET), it is important to calibrate the G factor in an experimental way.  However, if you look at relative Es at different conditions or samples within a same assay, you can theoretically estimate the G factor and use the same one for all your data analysis.
>
> Sheng
> Yuansheng Sun, Ph.D.
> W.M. Keck Center for Cellular Imaging
> http://www.kcci.virginia.edu/workshop/workshop2014/index.php
>
>
>
>
>
> On Tue, Apr 9, 2013 at 5:11 AM, Straatman, Kees R. (Dr.) < [hidden email]> wrote:
>
>> *****
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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
>>