AOBS spectrum

[Nuno Moreno]

Hi!

Does anyone know where can I find information about the Leica AOBS
spectral response? ...or from the AOTFs they use.

All the best,
--
Nuno Moreno, PhD
___________
Equipment Management Unit, Head
Instituto Gulbenkian de Ciência
Fundação Calouste Gulbenkian
phone   [phone]
fax     [phone]

[Ralf Zenke]

Dear Nuno,

The 'Product & Technology Guide' from Leica Confocal Systems contains some information on the AOBS. I found a diagram showing linear transmission of 92% for the full spectral range (450nm - 750nm). Excitation light will be blocked with a spectral width of approx. 1nm. The AOTF should have similar characteristics.

Hope that helps.

Nice regards,

Ralf

Ralf Zenke

Max Planck Institute of Biochemistry
Core Facility
Am Klopferspitz 18
DE-82152 Martinsried near Munich
GERMANY
Phone: (+49) (89) 8578 3798
Fax: (+49) (89) 8578 2847
www.biochem.mpg.de

Hi!

Does anyone know where can I find information about the Leica AOBS 
spectral response? ...or from the AOTFs they use.

All the best,

[Boswell, Carl A - (cboswell)]

I'd wager that there are many of us that would like to see that.
Good luck,

c

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Nuno Moreno" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, November 18, 2009 7:27 AM
Subject: AOBS spectrum

> Hi!
>
> Does anyone know where can I find information about the Leica AOBS
> spectral response? ...or from the AOTFs they use.
>
> All the best,
> --
> Nuno Moreno, PhD
> ___________
> Equipment Management Unit, Head
> Instituto Gulbenkian de Ciência
> Fundação Calouste Gulbenkian
> phone   [phone]
> fax     [phone]
>

[James Pawley]

>I'd wager that there are many of us that would like to see that.
>Good luck,
>
>c
>
>Carl A. Boswell, Ph.D.
>Molecular and Cellular Biology
>University of Arizona
>520-954-7053
>FAX [phone]
>----- Original Message ----- From: "Nuno Moreno" <[hidden email]>
>To: <[hidden email]>
>Sent: Wednesday, November 18, 2009 7:27 AM
>Subject: AOBS spectrum

Hi all,

A measured response of the Leica AOBS can be
found as Figure 3-23 on page 57 of "The Handbook".

I gather that actually measuring it was very difficult but it came from Leica.

Cheers,

Jim Pawley

               **********************************************
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.

>
>>Hi!
>>
>>Does anyone know where can I find information
>>about the Leica AOBS spectral response? ...or
>>from the AOTFs they use.
>>
>>All the best,
>>--
>>Nuno Moreno, PhD
>>___________
>>Equipment Management Unit, Head
>>Instituto Gulbenkian de Ciência
>>Fundação Calouste Gulbenkian
>>phone   [phone]
>>fax     [phone]

--

[Nuno Moreno]

Thank you Pawley,

I know that one but I wonder if there are other sources. I'm just as
asking this because if you measure the system performance from 400 to
700 it gives you a flat response. I was expecting it to behave in
accordance to a PMT profile.

Nuno Moreno, PhD
___________
Equipment Management Unit, Head
Instituto Gulbenkian de Ciência
Fundação Calouste Gulbenkian
phone   [phone]
fax     [phone]


James Pawley escreveu:

>> I'd wager that there are many of us that would like to see that.
>> Good luck,
>>
>> c
>>
>> Carl A. Boswell, Ph.D.
>> Molecular and Cellular Biology
>> University of Arizona
>> 520-954-7053
>> FAX [phone]
>> ----- Original Message ----- From: "Nuno Moreno"
>> <[hidden email]>
>> To: <[hidden email]>
>> Sent: Wednesday, November 18, 2009 7:27 AM
>> Subject: AOBS spectrum
>
>
> Hi all,
>
> A measured response of the Leica AOBS can be found as Figure 3-23 on
> page 57 of "The Handbook".
>
> I gather that actually measuring it was very difficult but it came from
> Leica.
>
> Cheers,
>
> Jim Pawley
>
>               **********************************************
> Prof. James B. Pawley,                                   Ph.  
> 608-263-3147 Room 223, Zoology Research
> Building,                              FAX [phone]
> 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.
>
>>
>>> Hi!
>>>
>>> Does anyone know where can I find information about the Leica AOBS
>>> spectral response? ...or from the AOTFs they use.
>>>
>>> All the best,
>>> --
>>> Nuno Moreno, PhD
>>> ___________
>>> Equipment Management Unit, Head
>>> Instituto Gulbenkian de Ciência
>>> Fundação Calouste Gulbenkian
>>> phone   [phone]
>>> fax     [phone]
>
>

[Guy Cox]

I suppose they build in a correction for the detector response - after all, it is supposed to be a spectrometer.

                                    Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nuno Moreno
Sent: Friday, 20 November 2009 3:38 AM
To: [hidden email]
Subject: Re: AOBS spectrum

Thank you Pawley,

I know that one but I wonder if there are other sources. I'm just as asking this because if you measure the system performance from 400 to 700 it gives you a flat response. I was expecting it to behave in accordance to a PMT profile.

Nuno Moreno, PhD
___________
Equipment Management Unit, Head
Instituto Gulbenkian de Ciência
Fundação Calouste Gulbenkian
phone   [phone]
fax     [phone]


James Pawley escreveu:

>> I'd wager that there are many of us that would like to see that.
>> Good luck,
>>
>> c
>>
>> Carl A. Boswell, Ph.D.
>> Molecular and Cellular Biology
>> University of Arizona
>> 520-954-7053
>> FAX [phone]
>> ----- Original Message ----- From: "Nuno Moreno"
>> <[hidden email]>
>> To: <[hidden email]>
>> Sent: Wednesday, November 18, 2009 7:27 AM
>> Subject: AOBS spectrum
>
>
> Hi all,
>
> A measured response of the Leica AOBS can be found as Figure 3-23 on
> page 57 of "The Handbook".
>
> I gather that actually measuring it was very difficult but it came
> from Leica.
>
> Cheers,
>
> Jim Pawley
>
>               **********************************************
> Prof. James B. Pawley,                                   Ph.  
> 608-263-3147 Room 223, Zoology Research
> Building,                              FAX [phone]
> 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.
>
>>
>>> Hi!
>>>
>>> Does anyone know where can I find information about the Leica AOBS
>>> spectral response? ...or from the AOTFs they use.
>>>
>>> All the best,
>>> --
>>> Nuno Moreno, PhD
>>> ___________
>>> Equipment Management Unit, Head
>>> Instituto Gulbenkian de Ciência
>>> Fundação Calouste Gulbenkian
>>> phone   [phone]
>>> fax     [phone]
>
>

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.707 / Virus Database: 270.14.73/2512 - Release Date: 11/19/09 06:41:00

[Koo, Lily (NIH/NIAID) [E]]

Hello list,

A while ago (2005 to be precise) there was a list exchange regarding the Forster distance of FRET pairs.  Iain Johnson from Molecular Probe (now Invitrogen) posted a link to his measured table of Forster distance for Alexa dyes.  Of course the link is no longer valid.  I am wondering if Iain could generously provide that information again?  Also, does anyone know of other existing tables for the other FRET pairs?  

Thanks,

Lily

[Johnson, Iain-2]

Apologies for the broken links.  The ones below are current:

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/tables/R0-values-for-some-Alexa-Fluor-dyes.html

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/tables/Ro-values-for-QSY-and-dabcyl-quenchers.html

For FPs there are some good literature sources including:

Anal Biochem. 2000 284(2):438-40.
Förster distances between green fluorescent protein pairs.
Patterson GH, Piston DW, Barisas BG. http://www.ncbi.nlm.nih.gov/sites/entrez/10964438

Iain

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Koo, Lily (NIH/NIAID) [E]
Sent: Thursday, November 19, 2009 2:41 PM
To: [hidden email]
Subject: FRET Forster distance - Iain Johnson's table and others

Hello list,

A while ago (2005 to be precise) there was a list exchange regarding the Forster distance of FRET pairs.  Iain Johnson from Molecular Probe (now Invitrogen) posted a link to his measured table of Forster distance for Alexa dyes.  Of course the link is no longer valid.  I am wondering if Iain could generously provide that information again?  Also, does anyone know of other existing tables for the other FRET pairs?  

Thanks,

Lily

[Kimmo K Tanhuanpaa]

On Thu, 19 Nov 2009, Koo, Lily (NIH/NIAID) [E] wrote:

Hi,

> A while ago (2005 to be precise) there was a list exchange regarding the
> Forster distance of FRET pairs.  Iain Johnson from Molecular Probe (now
> Invitrogen) posted a link to his measured table of Forster distance for
> Alexa dyes.  Of course the link is no longer valid.

You can find it in Molecular Probes Handbook, table 1.6:
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/tables/R0-values-for-some-Alexa-Fluor-dyes.html.html

Cheers,

Kimmo
_____________________________________________________________________
            Mr. Kimmo Tanhuanpää, PhD (Biochemistry)
                Head of Light Microscopy Unit,
        Institute of Biotechnology, University of Helsinki
            http://www.biocenter.helsinki.fi/bi/lmu/

[Nuno Moreno]

It is for me hard to understand why are they throwing away efficiency in
the blue/green range. In my head making it work as a spectrometer does
not worth it. I hope this is only true if we use the lambda mode!

Nuno Moreno, PhD
___________
Equipment Management Unit, Head
Instituto Gulbenkian de Ciência
Fundação Calouste Gulbenkian
phone   [phone]
fax     [phone]


Guy Cox escreveu:

> I suppose they build in a correction for the detector response - after all, it is supposed to be a spectrometer.
>
>                                     Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nuno Moreno
> Sent: Friday, 20 November 2009 3:38 AM
> To: [hidden email]
> Subject: Re: AOBS spectrum
>
> Thank you Pawley,
>
> I know that one but I wonder if there are other sources. I'm just as asking this because if you measure the system performance from 400 to 700 it gives you a flat response. I was expecting it to behave in accordance to a PMT profile.
>
> Nuno Moreno, PhD
> ___________
> Equipment Management Unit, Head
> Instituto Gulbenkian de Ciência
> Fundação Calouste Gulbenkian
> phone   [phone]
> fax     [phone]
>
>
> James Pawley escreveu:
>>> I'd wager that there are many of us that would like to see that.
>>> Good luck,
>>>
>>> c
>>>
>>> Carl A. Boswell, Ph.D.
>>> Molecular and Cellular Biology
>>> University of Arizona
>>> 520-954-7053
>>> FAX [phone]
>>> ----- Original Message ----- From: "Nuno Moreno"
>>> <[hidden email]>
>>> To: <[hidden email]>
>>> Sent: Wednesday, November 18, 2009 7:27 AM
>>> Subject: AOBS spectrum
>>
>> Hi all,
>>
>> A measured response of the Leica AOBS can be found as Figure 3-23 on
>> page 57 of "The Handbook".
>>
>> I gather that actually measuring it was very difficult but it came
>> from Leica.
>>
>> Cheers,
>>
>> Jim Pawley
>>
>>               **********************************************
>> Prof. James B. Pawley,                                   Ph.  
>> 608-263-3147 Room 223, Zoology Research
>> Building,                              FAX [phone]
>> 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.
>>
>>>> Hi!
>>>>
>>>> Does anyone know where can I find information about the Leica AOBS
>>>> spectral response? ...or from the AOTFs they use.
>>>>
>>>> All the best,
>>>> --
>>>> Nuno Moreno, PhD
>>>> ___________
>>>> Equipment Management Unit, Head
>>>> Instituto Gulbenkian de Ciência
>>>> Fundação Calouste Gulbenkian
>>>> phone   [phone]
>>>> fax     [phone]
>>
>
> No virus found in this incoming message.
> Checked by AVG - www.avg.com
> Version: 9.0.707 / Virus Database: 270.14.73/2512 - Release Date: 11/19/09 06:41:00
>

[George McNamara]

Hi Lily,

You can calculate Ro, as well as J* (spectral overlap integral - in
many ways more useful than Ro) using the spectra in the PubSpectra
xlsx file and the Szollosi&Horvath FRET Excel file, both in
http://www.sylvester.org/AICF/pubspectra.zip

I don't have the Excel matrices skill set to create a comprehensive
FRET donor vs acceptor table, but perhaps you or someone on the
listserv would like to do this - whoever does it could write the work
up for publication in Biotechniques, Nature Methods, etc.

George
p.s. there is also a nascent FRET calculator option at
http://www.mcb.arizona.edu/ipc/fret/index.html (3rd tab above the
graph box) - Carl Boswell at UA might benefit from a financial
donation to make the "Calculate FRET" button fully functional (or my
choice of Alexa Fluor 488 and 568 might not have QY or another variable).

p.p.s. Ro vs J* ...  Ro depends on quantum yield and somewhat on
refractive index which many papers do not mention. My understanding
is that the RI(s) in an ~1 um diameter sphere surrounding the
molecules is "sensed' (search Pubmed for Suhling refractive
index  for an entry point to this literature). RI 1.34 (water) or RI
1.5 (immersion oil? glass? solid protein?) which are clearly not
correct for cytosol (which is ~1.38 for mammalian cells). It would be
useful to define a normalized J* parameter that results in easy to
compare values (much like GM units (10^50 multiplier) puts
multiphoton absorption into a easy to read range, or QY * Extinction
coefficient / 1000 makes fluorophore efficiencies easier to read.

At 05:41 PM 11/19/2009, you wrote:

>Hello list,
>
>A while ago (2005 to be precise) there was a list exchange regarding
>the Forster distance of FRET pairs.  Iain Johnson from Molecular
>Probe (now Invitrogen) posted a link to his measured table of
>Forster distance for Alexa dyes.  Of course the link is no longer
>valid.  I am wondering if Iain could generously provide that
>information again?  Also, does anyone know of other existing tables
>for the other FRET pairs?
>
>Thanks,
>
>Lily

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+
spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

[Mark Cannell]

Hi All,

I must admit to being kind of bemused by this obsession with theoretical
FRET distances in cell experiments. In view of all the uncertainties in
dipole orientattion (etc.) I think that all you can really say is that
if FRET occurs the dipoles are very close together (a few nm). If it
changes, they are moving (translation and/or orientation) or the local
environment is changing (lipid<-->cytosol, another protein
entering/leaving) -and you really don't know which, of any of these, is
the cause. I think it very revealing when people talk about absolute
FRET distances and their changes when they really don't know what is
going on. Why do papers generally not talk about the possible change in
conformation/orientation of a protein or even that that
insertion/removal of a third protein may be responsible for a FRET change?

Cheers Mark

George McNamara wrote:

> Hi Lily,
>
> You can calculate Ro, as well as J* (spectral overlap integral - in
> many ways more useful than Ro) using the spectra in the PubSpectra
> xlsx file and the Szollosi&Horvath FRET Excel file, both in
> http://www.sylvester.org/AICF/pubspectra.zip
>
> I don't have the Excel matrices skill set to create a comprehensive
> FRET donor vs acceptor table, but perhaps you or someone on the
> listserv would like to do this - whoever does it could write the work
> up for publication in Biotechniques, Nature Methods, etc.
>
> George
> p.s. there is also a nascent FRET calculator option at
> http://www.mcb.arizona.edu/ipc/fret/index.html (3rd tab above the
> graph box) - Carl Boswell at UA might benefit from a financial
> donation to make the "Calculate FRET" button fully functional (or my
> choice of Alexa Fluor 488 and 568 might not have QY or another variable).
>
> p.p.s. Ro vs J* ...  Ro depends on quantum yield and somewhat on
> refractive index which many papers do not mention. My understanding is
> that the RI(s) in an ~1 um diameter sphere surrounding the molecules
> is "sensed' (search Pubmed for Suhling refractive index  for an entry
> point to this literature). RI 1.34 (water) or RI 1.5 (immersion oil?
> glass? solid protein?) which are clearly not correct for cytosol
> (which is ~1.38 for mammalian cells). It would be useful to define a
> normalized J* parameter that results in easy to compare values (much
> like GM units (10^50 multiplier) puts multiphoton absorption into a
> easy to read range, or QY * Extinction coefficient / 1000 makes
> fluorophore efficiencies easier to read.
>
> At 05:41 PM 11/19/2009, you wrote:
>> Hello list,
>>
>> A while ago (2005 to be precise) there was a list exchange regarding
>> the Forster distance of FRET pairs.  Iain Johnson from Molecular
>> Probe (now Invitrogen) posted a link to his measured table of Forster
>> distance for Alexa dyes.  Of course the link is no longer valid.  I
>> am wondering if Iain could generously provide that information
>> again?  Also, does anyone know of other existing tables for the other
>> FRET pairs?
>>
>> Thanks,
>>
>> Lily
>
>
>
>
>
>
>
> George McNamara, Ph.D.
> Image Core Manager
> Analytical Imaging Core Facility
> University of Miami, Miller School of Medicine
> Miami, FL 33136
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
> http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra
> .xlsx file is in the zip file)
> http://home.earthlink.net/~geomcnamara

[Vitaly Boyko]

Well, as most of the fluorescent proteins are either dimers or worse upon desired over expression, "for easy going" (sometimes even with the letter "m" in front of). The latter fact would yield in positive but often "wishful" FRET signal, especially when carefully limited number of controls have been designed to be " biophysically smart". Therefore, all the discussions around the Ro or J seem to be redundunt... You might recollect that the famous textbook experiments have been performed with free, soluble donor-acceptor pairs at concentrations far above physiologically acceptable limits.

 

...my two cents...

 

Vitaly

---

From: Mark Cannell <[hidden email]>
To: [hidden email]
Sent: Sun, November 22, 2009 1:51:19 PM
Subject: Re: FRET Forster distance - Iain Johnson's table and others

Hi All,

I must admit to being kind of bemused by this obsession with theoretical FRET distances in cell experiments. In view of all the uncertainties in dipole orientattion (etc.) I think that all you can really say is that if FRET occurs the dipoles are very close together (a few nm). If it changes, they are moving (translation and/or orientation) or the local environment is changing (lipid<-->cytosol, another protein entering/leaving) -and you really don't know which, of any of these, is the cause. I think it very revealing when people talk about absolute FRET distances and their changes when they really don't know what is going on. Why do papers generally not talk about the possible change in conformation/orientation of a protein or even that that insertion/removal of a third protein may be responsible for a FRET change?

Cheers Mark

George McNamara wrote:

> Hi Lily,
>
> You can calculate Ro, as well as J* (spectral overlap integral - in many ways more useful than Ro) using the spectra in the PubSpectra xlsx file and the Szollosi&Horvath FRET Excel file, both in http://www.sylvester.org/AICF/pubspectra.zip
>
> I don't have the Excel matrices skill set to create a comprehensive FRET donor vs acceptor table, but perhaps you or someone on the listserv would like to do this - whoever does it could write the work up for publication in Biotechniques, Nature Methods, etc.
>
> George
> p.s. there is also a nascent FRET calculator option at http://www.mcb.arizona.edu/ipc/fret/index.html (3rd tab above the graph box) - Carl Boswell at UA might benefit from a financial donation to make the "Calculate FRET" button fully functional (or my choice of Alexa Fluor 488 and 568 might not have QY or another variable).
>
> p.p.s. Ro vs J* ...  Ro depends on quantum yield and somewhat on refractive index which many papers do not mention. My understanding is that the RI(s) in an ~1 um diameter sphere surrounding the molecules is "sensed' (search Pubmed for Suhling refractive index  for an entry point to this literature). RI 1.34 (water) or RI 1.5 (immersion oil? glass? solid protein?) which are clearly not correct for cytosol (which is ~1.38 for mammalian cells). It would be useful to define a normalized J* parameter that results in easy to compare values (much like GM units (10^50 multiplier) puts multiphoton absorption into a easy to read range, or QY * Extinction coefficient / 1000 makes fluorophore efficiencies easier to read.
>
> At 05:41 PM 11/19/2009, you wrote:
>> Hello list,
>>
>> A while ago (2005 to be precise) there was a list exchange regarding the Forster distance of FRET pairs.  Iain Johnson from Molecular Probe (now Invitrogen) posted a link to his measured table of Forster distance for Alexa dyes.  Of course the link is no longer valid.  I am wondering if Iain could generously provide that information again?  Also, does anyone know of other existing tables for the other FRET pairs?
>>
>> Thanks,
>>
>> Lily
>
>
>
>
>
>
>
> George McNamara, Ph.D.
> Image Core Manager
> Analytical Imaging Core Facility
> University of Miami, Miller School of Medicine
> Miami, FL 33136
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
> http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
> http://home.earthlink.net/~geomcnamara