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Fwd: FRET - PMT Spectral Sensitivity

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Daniel James White

Fwd: FRET - PMT Spectral Sensitivity

Hi Jacui,

For sure it does matter, and to find out those cirves,
yiou need to ask the supplier of your microscope.

I'm guessing its a confocal,
and then likely a Zeiss, Olympus, Nikon or Leica.

Ask your local rep for the spec sheet for the PMTs in the instrument
you have,
and make sure that contains the spectra response of the PMT.

cheers

Dan

Begin forwarded message:

> Date: Fri, 12 Mar 2010 17:11:58 +1300
> From: Jacqui Ross <[hidden email]>
> Subject: FW: FRET - PMT Spectral Sensitivity
>
> Dear All,
>
> One of our researchers is using the pFRET plugin for ImageJ. He has a
> query regarding the PMT Spectral Sensitivity as per the email below.
> I'm
> not familiar with this plugin.
>
> Is anyone able to answer this question? I will forward any replies to
> Pablo.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross=20
>
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit=20
> School of Medical Sciences=20
> Faculty of Medical & Health Sciences=20
> The University of Auckland=20
> Private Bag 92019=20
> Auckland, NEW ZEALAND=20
>
> Tel: 64 9 373 7599 Ext 87438=20
> Fax: 64 9 373 7484=20
>
> http://www.fmhs.auckland.ac.nz/sms/biru/
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ]
> On Behalf Of Pablo German
> Sent: Friday, 12 March 2010 4:17 p.m.
> 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
>
> --=20
> Pablo German
> PhD Candidate
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: (09) 925-7107
> Mobile: 0210459406

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
+49 (0)351 210 1078 (Fax MPI-CBG LMF)

http://www.bioimagexd.net BioImageXD
http://pacific.mpi-cbg.de Fiji - is just ImageJ (Batteries Included)
http://www.chalkie.org.uk Dan's Homepages
https://ifn.mpi-cbg.de Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

James Pawley

Re: Fwd: FRET - PMT Spectral Sensitivity

>Hi Jacui,
>
>For sure it does matter, and to find out those cirves,
>yiou need to ask the supplier of your microscope.
>
>I'm guessing its a confocal,
>and then likely a Zeiss, Olympus, Nikon or Leica.
>
>Ask your local rep for the spec sheet for the PMTs in the instrument you have,
>and make sure that contains the spectra response of the PMT.
>
>cheers
>
>Dan
>

Hi Jacui,

I agree with Dan: Spectral sensitivity is very important. And indeed
the spec sheets may be of some help, especially on side-window PMTs.
However, on end-window PMTs there is a lot more tube-to-tube
variation and there is even variation across the sensitive area of
the tube. This is often because the almost-transparent layer of
photocathode material is not evaporated onto the inside of the window
until after the tube has been pumped down and sealed off. Given the
geometry, it is hard to ensure that this film has a uniform thickness
and, all other things being equal, thicker areas will have relatively
better red sensitivity (vs. blue) than thinner areas.

So it might be even better if you can arrange some sort of direct
measurement made from standard specimens composed of solution of
known concentration of the relevant dyes.

On the other hand, quantitative FRET of any kind is "very
challenging", so don't let the perfect be the enemy of the
"good-enough."

Cheers,

Jim P.

**********************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2010
"If it ain't diffraction, it must be statistics." Anon.

>
>Begin forwarded message:
>
>>Date: Fri, 12 Mar 2010 17:11:58 +1300
>>From: Jacqui Ross <[hidden email]>
>>Subject: FW: FRET - PMT Spectral Sensitivity
>>
>>Dear All,
>>
>>One of our researchers is using the pFRET plugin for ImageJ. He has a
>>query regarding the PMT Spectral Sensitivity as per the email below. I'm
>>not familiar with this plugin.
>>
>>Is anyone able to answer this question? I will forward any replies to
>>Pablo.
>>
>>Kind regards,
>>
>>Jacqui
>>
>>Jacqueline Ross=20
>>
>>Biomedical Imaging Microscopist
>>Biomedical Imaging Research Unit=20
>>School of Medical Sciences=20
>>Faculty of Medical & Health Sciences=20
>>The University of Auckland=20
>>Private Bag 92019=20
>>Auckland, NEW ZEALAND=20
>>
>>Tel: 64 9 373 7599 Ext 87438=20
>>Fax: 64 9 373 7484=20
>>
>>http://www.fmhs.auckland.ac.nz/sms/biru/
>>
>>
>>-----Original Message-----
>>From: Confocal Microscopy List [mailto:[hidden email]]
>>On Behalf Of Pablo German
>>Sent: Friday, 12 March 2010 4:17 p.m.
>>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
>>
>>--=20
>>Pablo German
>>PhD Candidate
>>
>>Plant and Food Research
>>Private Bag 92169
>>Auckland Mail Centre
>>Auckland 1142
>>New Zealand
>>DDI: (09) 925-7107
>>Mobile: 0210459406
>
>Dr. Daniel James White BSc. (Hons.) PhD
>Senior Microscopist / Image Visualisation, Processing and Analysis
>Light Microscopy and Image Processing Facilities
>Max Planck Institute of Molecular Cell Biology and Genetics
>Pfotenhauerstrasse 108
>01307 DRESDEN
>Germany
>
>+49 (0)15114966933 (German Mobile)
>+49 (0)351 210 2627 (Work phone at MPI-CBG)
>+49 (0)351 210 1078 (Fax MPI-CBG LMF)
>
>http://www.bioimagexd.net BioImageXD
>http://pacific.mpi-cbg.de Fiji - is just ImageJ
>(Batteries Included)
>http://www.chalkie.org.uk Dan's Homepages
>https://ifn.mpi-cbg.de Dresden Imaging Facility Network
>dan (at) chalkie.org.uk
>( white (at) mpi-cbg.de )

--
**********************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2010
"If it ain't diffraction, it must be statistics." Anon.

Jacqueline Ross

Re: Fwd: FRET - PMT Spectral Sensitivity

Hi Jim & Yuansheng,

Thanks to both of you for that information, which I am sure Pablo has
also read. I think I do have some specifications for the PMTs for the
system but if not, we will contact Leica. The system Pablo is using is a
Leica TCS SP2.

We also have the book that Yuansheng Sun mentioned so I have let Pablo
know that.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: 64 9 373 7599 Ext 87438
Fax: 64 9 373 7484

http://www.fmhs.auckland.ac.nz/sms/biru/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of James Pawley
Sent: Saturday, 13 March 2010 10:51 a.m.
To: [hidden email]
Subject: Re: Fwd: FRET - PMT Spectral Sensitivity

>Hi Jacui,
>
>For sure it does matter, and to find out those cirves,
>yiou need to ask the supplier of your microscope.
>
>I'm guessing its a confocal,
>and then likely a Zeiss, Olympus, Nikon or Leica.
>
>Ask your local rep for the spec sheet for the PMTs in the instrument
you have,
>and make sure that contains the spectra response of the PMT.
>
>cheers
>
>Dan
>

Hi Jacui,

I agree with Dan: Spectral sensitivity is very important. And indeed
the spec sheets may be of some help, especially on side-window PMTs.
However, on end-window PMTs there is a lot more tube-to-tube
variation and there is even variation across the sensitive area of
the tube. This is often because the almost-transparent layer of
photocathode material is not evaporated onto the inside of the window
until after the tube has been pumped down and sealed off. Given the
geometry, it is hard to ensure that this film has a uniform thickness
and, all other things being equal, thicker areas will have relatively
better red sensitivity (vs. blue) than thinner areas.

So it might be even better if you can arrange some sort of direct
measurement made from standard specimens composed of solution of
known concentration of the relevant dyes.

On the other hand, quantitative FRET of any kind is "very
challenging", so don't let the perfect be the enemy of the
"good-enough."

Cheers,

Jim P.

**********************************************
Prof. James B. Pawley, Ph.
608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver
Canada
Info: http://www.3dcourse.ubc.ca/ Applications due by March
15, 2010
"If it ain't diffraction, it must be statistics." Anon.

I'm

>>not familiar with this plugin.
>>
>>Is anyone able to answer this question? I will forward any replies to
>>Pablo.
>>
>>Kind regards,
>>
>>Jacqui
>>
>>Jacqueline Ross=20
>>
>>Biomedical Imaging Microscopist
>>Biomedical Imaging Research Unit=20
>>School of Medical Sciences=20
>>Faculty of Medical & Health Sciences=20
>>The University of Auckland=20
>>Private Bag 92019=20
>>Auckland, NEW ZEALAND=20
>>
>>Tel: 64 9 373 7599 Ext 87438=20
>>Fax: 64 9 373 7484=20
>>
>>http://www.fmhs.auckland.ac.nz/sms/biru/
>>
>>
>>-----Original Message-----
>>From: Confocal Microscopy List

[mailto:[hidden email]]

>>On Behalf Of Pablo German
>>Sent: Friday, 12 March 2010 4:17 p.m.
>>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
>>
>>--=20
>>Pablo German
>>PhD Candidate
>>
>>Plant and Food Research
>>Private Bag 92169
>>Auckland Mail Centre
>>Auckland 1142
>>New Zealand
>>DDI: (09) 925-7107
>>Mobile: 0210459406
>
>Dr. Daniel James White BSc. (Hons.) PhD
>Senior Microscopist / Image Visualisation, Processing and Analysis
>Light Microscopy and Image Processing Facilities
>Max Planck Institute of Molecular Cell Biology and Genetics
>Pfotenhauerstrasse 108
>01307 DRESDEN
>Germany
>
>+49 (0)15114966933 (German Mobile)
>+49 (0)351 210 2627 (Work phone at MPI-CBG)
>+49 (0)351 210 1078 (Fax MPI-CBG LMF)
>
>http://www.bioimagexd.net BioImageXD
>http://pacific.mpi-cbg.de Fiji - is just ImageJ
>(Batteries Included)
>http://www.chalkie.org.uk Dan's Homepages
>https://ifn.mpi-cbg.de Dresden Imaging Facility Network
>dan (at) chalkie.org.uk
>( white (at) mpi-cbg.de )

--
**********************************************
Prof. James B. Pawley, Ph.
608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver
Canada
Info: http://www.3dcourse.ubc.ca/ Applications due by March
15, 2010
"If it ain't diffraction, it must be statistics." Anon.