FRET - PMT Spectral Sensitivity

[Pablo German]

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: [phone]
Mobile: [phone]

[leoncio vergara]

... the quantum efficiency of the detector versus wavelenght is something you would need to get from the manufacturer... unfortunately this may be a proprietary piece of information in most cases...

you need this information to calculate the FRET efficiency...

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Pablo German [[hidden email]]
Sent: Thursday, March 11, 2010 9:17 PM
To: [hidden email]
Subject: FRET - PMT Spectral Sensitivity

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: [phone]
Mobile: [phone]

[yuansheng sun]

Hi Pablo,

 

My name is Yuansheng Sun, working for Dr. Periasamy in the KCCI at UVA. I am currently maintaining the PFRET software.

 

If you use a CCD camera, which should usually give the same quantum efficiency for the eCFP and eYFP emission wavelengths, then you just leave both sensitivities as 1:1. If the PMT detectors are used, the sensitivities for eCFP and eYFP could be different, depending on the PMTs. You should be able to get the PMT quantum efficiency graph from where you bought the system or the PMT. Based on the graph, you can obtain the sensitivities at the peak emission wavelengths of eCFP and eYFP.

 

Details on why we need to consider the difference between the detector sensitivities for donor and acceptor are explained in Chapter 7 of Molecular Imaging, FRET Microscopy and Spectroscopy, edited by Ammasi Periasamy and Richard N. Day.

 

sheng

 

On Thu, Mar 11, 2010 at 10:17 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: [phone]
Mobile: [phone]

[Steve Ruzin-2]

Sure, I'll be there, 

Steve...

On Mar 11, 2010, at 7:59 PM, yuansheng sun wrote:

Hi Pablo,

 

My name is Yuansheng Sun, working for Dr. Periasamy in the KCCI at UVA. I am currently maintaining the PFRET software.

 

If you use a CCD camera, which should usually give the same quantum efficiency for the eCFP and eYFP emission wavelengths, then you just leave both sensitivities as 1:1. If the PMT detectors are used, the sensitivities for eCFP and eYFP could be different, depending on the PMTs. You should be able to get the PMT quantum efficiency graph from where you bought the system or the PMT. Based on the graph, you can obtain the sensitivities at the peak emission wavelengths of eCFP and eYFP.

 

Details on why we need to consider the difference between the detector sensitivities for donor and acceptor are explained in Chapter 7 of Molecular Imaging, FRET Microscopy and Spectroscopy, edited by Ammasi Periasamy and Richard N. Day.

 

sheng

 

On Thu, Mar 11, 2010 at 10:17 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: [phone]
Mobile: [phone]

__________________________________
Steven E Ruzin, PhD
Biological Imaging Facility
381 Koshland Hall
University of California
Berkeley, CA 94720-3102
510-642-6602
http://microscopy.berkeley.edu
http://golubcollection.berkeley.edu

[yuansheng sun]

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: [phone]
> Mobile: [phone]

[Pablo German]

Thanks Yuansheng,

Very comprehensive answer! I have been given the sensitivities of the
PMTs I'm using. On the other hand, I am not so concerned with absolute
efficiencies but relative efficiencies between different samples and
as you said, it's not so important.

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.

Regards,
Pablo



On 16 March 2010 11:32, yuansheng sun <[hidden email]> wrote:

> 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: [phone]
>> Mobile: [phone]
>

--
Pablo German

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]