http://confocal-microscopy-list.275.s1.nabble.com/FRET-PMT-Spectral-Sensitivity-tp4720210p4741246.html
PMTs I'm using. On the other hand, I am not so concerned with absolute
Yes, I was at the workshop last year. I'll send an email for an
updated copy of the PFRET software. Hope the workshop went well.
> 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
>>
>> Plant and Food Research
>> Private Bag 92169
>> Auckland Mail Centre
>> Auckland 1142
>> New Zealand
>> DDI: (09) 925-7107
>> Mobile: 0210459406
>