Re: Acceptor Photobleaching vs. Sensitized Emission FRET results

Posted by yuansheng sun on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p4830788.html

Dear Pablo,

This is sheng, working at the Keck center, UVA. I think we have
contacted earlier for the new pFRET software. Here are my comments for
this topic:

I would not be surprised to see different FRET efficiencies for
different Donor : Acceptor ratios or different Acceptor levels. That
is actually a valuable indication for the random association or the
cluster assembly. I recommend you look at the following paper -
Biophys. J. Vol 85, Issue 1, 559-571 (2003).
If you write me an email ([hidden email]), I can send you a
couple of nice PPTs on this topic.

I like your strategy - measuring FRET of a same system in different
ways (SE vs. AP). If possible, I would like to try FLIM as well since
lifetime is independent of fluorophore concentration, only if
possible. We have to work with what we have. I would not make the
decision to accept AP and reject SE, because I do not see a reason why
AP can give you more accurate (quantitative) results than SE, if your
experiments were done properly.

There are actually some potential issues you may check for using AP. I
assume your measurements were done with live cells.

1. Check if the donor is also bleached during the photobleaching
process. Use the donor-alone specimen to check. The apFRET plugin in
the new pFRET software allows you correct for this issue.

2. Check if the acceptor is completely bleached. Take the pre- and
post- acceptor images. Your FRET efficiency is certainly influenced by
the left acceptor amount after photobleaching. The apFRET plugin in
the new pFRET software allows you address this issue.

3. Check if there is any cellular movement or focus change. Overlay
pre- and post- images in two different colors to see if you will have
a perfect overlay. If not, I suggest you run AP with fixed cells to
see you will also have homogeneous FRET efficiencies.

Please shoot me an email if you need help using the pFRET software to
check the issues mentioned above. Good luck.

Best regards,
sheng




On Tue, Mar 30, 2010 at 11:53 PM, Pablo German
<[hidden email]> wrote:

> Dear list members,
>
> I have been doing some FRET microscopy experiments on a
> 7-TransMembrane domain receptor tagged with either eCFP/eYFP at the
> different intra-cellular loops (ICL1, ICL2, and ICL3). I have tried
> the 9 different combinations (e.g ICL1-YFP + ICL1-CFP, ICL1-YFP +
> ICL2-CFP, etc) to see if I could detect any difference in FRET
> efficiency.
>
> I have anlyzed the images by both Sensitized Emission and Acceptor
> Photobleaching using the pFRET plugin on ImageJ developed at KCCI-UVa.
> The problem is the following: the results using Sensitized Emission
> give me significant differences between the different pairs but the
> results using APB give me no differences (all about 25% efficiency).
>
> I have the feeling that I should trust APB more than SE. I have
> noticed that, when using SE, the higher the difference in intensity
> between YFP and CFP, the higher the FRET efficiency.
>
> Has anyone had a similar experience? Which method of analysis should I trust?
>
> Regards,
> Pablo
>
> --
> Pablo German
> PhD Candidate
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
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> Mobile: [phone]
>