Posted by
yuansheng sun on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FRET-PMT-Spectral-Sensitivity-tp4720210p4740340.html
Hi Pablo,
This is Yuansheng Sun again. We just had our 9th annual FRET workshop.
It ended on Saturday night. Now, I have time to write more on your
questions.
>> Where can I find PMT sensitivities for different wavelengths?
I think I got this part right in my earlier email. I agree with Dan
and James. No matter what, it is better to have an idea about your
detector quantum efficiency, especially for quantitative FRET imaging.
Sometimes, you do not see FRET signals you expect, maybe because the
detector cannot pick up signals at the acceptor emission wavelength
nicely. Here are some example numbers: The PMT in one of our confocal
systems shows the the quantum efficiencies of ~28% at 477 nm (eCFP)
and ~25% at 530 nm (eYFP). The difference (28:25) is quite small. The
PMT on another (old) confocal we have gives ~12% (eCFP) and ~8%
(eYFP). There is a 33% (12:8) difference, which is not trivial.
>> Is it important?
I had this question many times in the FRET workshop. It is difficult
to give an explicit answer. It depends on what you are looking at. In
sensitized FRET microscopy, FRET efficiency is calculated from the
quenched donor and the FRET signals, which are measured in the donor
channel and the FRET channel (after removing spectral bleedthroughs),
respectively. To link the same intensities obtained in the two
channels in the sense of same 'energy', we have to introduce a
"correction factor", which depends on the quantum yields of the donor
and acceptor fluorophores and also the instrument detection
efficiencies for them. It is very difficult to accurately calibrate
them, for the following reasons.
The donor (or acceptor) fluorophore quantum yield refers to the
quantum yield of the conjugated dye in real specimens, which can be
very different than that measured from purified dyes. Measuring the
quantum yield of CFP expressed in live cells can be very challenging,
and probably requires the lifetime work. On the other hand, depending
on the instrument setup, the detection efficiency for the donor or the
acceptor may involve other factors (such as the optical transmission
efficiency in the light pathway to each channel) besides the detector
sensitivity for each fluorophore. Methods were published for measuring
the correction factor - to list a few:
A. Hoppe, K. Christensen and J.A. Swanson, "Fluorescence resonance
energy transfer-based stoichiometry in living cells," Biophys J 83(6),
3652-64 (2002).
T. Zal and N.R. Gascoigne, "Photobleaching-corrected FRET efficiency
imaging of live cells," Biophys J 86(6), 3923-39 (2004).
H. Chen, H. L. Puhl 3rd, S. V. Koushik, S. S. Vogel and S. R. Ikeda,
"Measurement of FRET Efficiency and Ratio of Donor to Acceptor
Concetration in Living Cells," Biophysical Journal 91(5): L39-L41
(2006).
All three methods used a reference specimen with Donor:Acceptor=1:1.
Hoppe method took the lifetime measurement as a reference. Zal method
employed the acceptor photobleaching technique. Chen method did not
require a second technique but used two reference specimens - both
have Donor:Acceptor as 1:1 but give different FRET efficiencies. I
cannot say which method is suitable for you until you give me some
details about your experiments. But I definitely recommend you read
those papers, which are all written very clearly. We are planning to
do some work to compare these calibrations.
Now, let's come to the final point. Is it important to calibrate the
correction factor? Obviously, the work for an accurate calibration is
not trivial. In my opinion, the calibration needs to be done if you
need to know the exact apparent FRET efficiency or the exact
Donor:Acceptor ratio. However, if you want to compare your
FRET-positive (or high-FRET) and FRET-negative (or low-FRET) specimens
based on the relative apparent FRET efficiencies vs. relative
Donor:Acceptor ratios, and run your FRET experiments on the same
instrument with the same settings, I do not see a real importance for
the detailed calibration.
If you are one of our previous FRET workshop participants, please
contact us (
[hidden email]) and we will send you an updated PFRET
software. I would like to thank all of our participants for their
valuable comments and suggestions, which essentially drive us to
improve the PFRET software every year.
Best regards,
sheng
On Thu, Mar 11, 2010 at 11:17 PM, Pablo German
<
[hidden email]> wrote:
>
> Hi,
>
> I'm analysing FRET images using the pFRET plugin of ImageJ developed
> at Universit of Virginia.
>
> When setting the parameters it asks me for the PMT Spectral
> Sensitivity for donor and acceptor. I'm using eCFP and eYFP. Where can
> I find these values? Is it important?
>
> Regards,
> Pablo
>
> --
> Pablo German
> PhD Candidate
>
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