Zeiss or Olympus

[Vladimir Ghukasyan-2]

Dear List Members,

We're making a selection between Zeiss 710 and Olympus FV1000 for the
multiphoton imaging.
I would be thankful for all user experience cases and suggestions on
this matter.

Thank you in advance.
With regards,
Vladimir

---------------------------------
Vladimir Ghukasyan
Confocal and Multiphoton Imaging Core,
Neuroscience Research Center
University of North Carolina
115 Mason Farm Rd., Bld. 245, 7 Fl.
Chapel Hill, NC 27599-7250
Tel.: [phone]

[Stephan Speier]

Dear Vladimir,

We tested the Zeiss LSM710 and the Olympus FV1000 regarding confocal
and multiphoton imaging and had already decided for the FV1000 when
Zeiss released the LSM780.
After testing the LSM780 we changed our mind and decided for Zeiss.
For our purposes the LSM780 has exactly the improvements over the 710,
we were looking for and therefore beat the Olympus.

Best,
Stephan


----- Original Message -----
From: Vladimir Gukassyan <[hidden email]>
Date: Tuesday, March 9, 2010 1:18 pm
Subject: Zeiss or Olympus
To: [hidden email]

> Dear List Members,
>
> We're making a selection between Zeiss 710 and Olympus FV1000 for the
> multiphoton imaging.
> I would be thankful for all user experience cases and suggestions on
> this matter.
>
> Thank you in advance.
> With regards,
> Vladimir
>
> ---------------------------------
> Vladimir Ghukasyan
> Confocal and Multiphoton Imaging Core,
> Neuroscience Research Center
> University of North Carolina
> 115 Mason Farm Rd., Bld. 245, 7 Fl.
> Chapel Hill, NC 27599-7250
> Tel.: [phone]
>

[Vladimir Ghukasyan-2]

Dear Stephan,

Thank you for your reply.
Could you also mention what were the advantages of the 780 over 710?
If not too much trouble, could you please also write a couple of words
on why you initially preferred FV1000? Did the systems differ in
penetration depth?

Thank you in advance,
Vladimir


On Tue, Mar 9, 2010 at 8:10 AM, Stephan Speier <[hidden email]> wrote:

> Dear Vladimir,
>
> We tested the Zeiss LSM710 and the Olympus FV1000 regarding confocal
> and multiphoton imaging and had already decided for the FV1000 when
> Zeiss released the LSM780.
> After testing the LSM780 we changed our mind and decided for Zeiss.
> For our purposes the LSM780 has exactly the improvements over the 710,
> we were looking for and therefore beat the Olympus.
>
> Best,
> Stephan
>
>
> ----- Original Message -----
> From: Vladimir Gukassyan <[hidden email]>
> Date: Tuesday, March 9, 2010 1:18 pm
> Subject: Zeiss or Olympus
> To: [hidden email]
>
>> Dear List Members,
>>
>> We're making a selection between Zeiss 710 and Olympus FV1000 for the
>> multiphoton imaging.
>> I would be thankful for all user experience cases and suggestions on
>> this matter.
>>
>> Thank you in advance.
>> With regards,
>> Vladimir
>>
>> ---------------------------------
>> Vladimir Ghukasyan
>> Confocal and Multiphoton Imaging Core,
>> Neuroscience Research Center
>> University of North Carolina
>> 115 Mason Farm Rd., Bld. 245, 7 Fl.
>> Chapel Hill, NC 27599-7250
>> Tel.: [phone]
>>
>

[RICHARD BURRY]

Vladimir and Stephan

 

The Zeiss 780 is an upgrade for the single photon.  The big issues for me with the Zeiss and Olympus multiphoton are the detector dynamic range, and detector sensitivity.  Extremely sensitive detectors can have a limited dynamic range.  I would be interested in hearing from someone who had recently tested not only the sensitivity of these two multiphotons, but also the dynamic range in an image collected a moderate to low fluorescence levels.  My evaluation a year ago was that the Olympus multiphoton had the better combination of sensitivity and dynamic range.

 

Dick Burry

----- Original Message -----
From: Stephan Speier <[hidden email]>
Date: Tuesday, March 9, 2010 8:10 am
Subject: Re: Zeiss or Olympus
To: [hidden email]

> Dear Vladimir,
>
> We tested the Zeiss LSM710 and the Olympus FV1000 regarding
> confocal
> and multiphoton imaging and had already decided for the FV1000
> when
> Zeiss released the LSM780.
> After testing the LSM780 we changed our mind and decided for Zeiss.
> For our purposes the LSM780 has exactly the improvements over
> the 710,
> we were looking for and therefore beat the Olympus.
>
> Best,
> Stephan
>
>
> ----- Original Message -----
> From: Vladimir Gukassyan <[hidden email]>
> Date: Tuesday, March 9, 2010 1:18 pm
> Subject: Zeiss or Olympus
> To: [hidden email]
>
> > Dear List Members,
> >
> > We're making a selection between Zeiss 710 and Olympus FV1000
> for the
> > multiphoton imaging.
> > I would be thankful for all user experience cases and
> suggestions on
> > this matter.
> >
> > Thank you in advance.
> > With regards,
> > Vladimir
> >
> > ---------------------------------
> > Vladimir Ghukasyan
> > Confocal and Multiphoton Imaging Core,
> > Neuroscience Research Center
> > University of North Carolina
> > 115 Mason Farm Rd., Bld. 245, 7 Fl.
> > Chapel Hill, NC 27599-7250
> > Tel.: [phone]
> >
>
>
> --
> BEGIN-ANTISPAM-VOTING-LINKS
> ------------------------------------------------------
>
> Teach CanIt if this mail (ID 1010315472) is spam:
> Spam:       
> https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=sNot spam:    https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=n
> Forget vote:
> https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=f---
> ---------------------------------------------------
> END-ANTISPAM-VOTING-LINKS
>

[Craig Brideau]

Another factor to consider is the service and support level each
company can provide at your location.  I've heard good things and bad
things about all the major companies; much of it depends on your LOCAL
service people for each company.

Craig


On Tue, Mar 9, 2010 at 7:59 AM, RICHARD BURRY <[hidden email]> wrote:

> Vladimir and Stephan
>
>
>
> The Zeiss 780 is an upgrade for the single photon.  The big issues for me
> with the Zeiss and Olympus multiphoton are the detector dynamic range, and
> detector sensitivity.  Extremely sensitive detectors can have a limited
> dynamic range.  I would be interested in hearing from someone who had
> recently tested not only the sensitivity of these two multiphotons, but also
> the dynamic range in an image collected a moderate to low fluorescence
> levels.  My evaluation a year ago was that the Olympus multiphoton had the
> better combination of sensitivity and dynamic range.
>
>
>
> Dick Burry
>
> ----- Original Message -----
> From: Stephan Speier <[hidden email]>
> Date: Tuesday, March 9, 2010 8:10 am
> Subject: Re: Zeiss or Olympus
> To: [hidden email]
>
>> Dear Vladimir,
>>
>> We tested the Zeiss LSM710 and the Olympus FV1000 regarding
>> confocal
>> and multiphoton imaging and had already decided for the FV1000
>> when
>> Zeiss released the LSM780.
>> After testing the LSM780 we changed our mind and decided for Zeiss.
>> For our purposes the LSM780 has exactly the improvements over
>> the 710,
>> we were looking for and therefore beat the Olympus.
>>
>> Best,
>> Stephan
>>
>>
>> ----- Original Message -----
>> From: Vladimir Gukassyan <[hidden email]>
>> Date: Tuesday, March 9, 2010 1:18 pm
>> Subject: Zeiss or Olympus
>> To: [hidden email]
>>
>> > Dear List Members,
>> >
>> > We're making a selection between Zeiss 710 and Olympus FV1000
>> for the
>> > multiphoton imaging.
>> > I would be thankful for all user experience cases and
>> suggestions on
>> > this matter.
>> >
>> > Thank you in advance.
>> > With regards,
>> > Vladimir
>> >
>> > ---------------------------------
>> > Vladimir Ghukasyan
>> > Confocal and Multiphoton Imaging Core,
>> > Neuroscience Research Center
>> > University of North Carolina
>> > 115 Mason Farm Rd., Bld. 245, 7 Fl.
>> > Chapel Hill, NC 27599-7250
>> > Tel.: [phone]
>> >
>>
>>
>> --
>> BEGIN-ANTISPAM-VOTING-LINKS
>> ------------------------------------------------------
>>
>> Teach CanIt if this mail (ID 1010315472) is spam:
>> Spam:
>> https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=sNot spam:
>> https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=n
>> Forget vote:
>> https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=f---
>> ---------------------------------------------------
>> END-ANTISPAM-VOTING-LINKS
>>

[Mark Cannell]

Hi RICHARD

As far as I know, for PMT based systems the dynamic range does not
depend on PMT sensitivity (which is determined by the photocathode) but
on gain (=PMT HV) and A/D settings.

Cheers Mark

BURRY wrote:

>
> Vladimir and Stephan
>
>  
>
> The Zeiss 780 is an upgrade for the single photon.  The big issues for
> me with the Zeiss and Olympus multiphoton are the detector dynamic
> range, and detector sensitivity.  Extremely sensitive detectors can
> have a limited dynamic range.  I would be interested in hearing from
> someone who had recently tested not only the sensitivity of these two
> multiphotons, but also the dynamic range in an image collected a
> moderate to low fluorescence levels.  My evaluation a year ago was
> that the Olympus multiphoton had the better combination of sensitivity
> and dynamic range.
>
>  
>
> Dick Burry
>
>
> ----- Original Message -----
> From: Stephan Speier <[hidden email]>
> Date: Tuesday, March 9, 2010 8:10 am
> Subject: Re: Zeiss or Olympus
> To: [hidden email]
>
> > Dear Vladimir,
> >
> > We tested the Zeiss LSM710 and the Olympus FV1000 regarding
> > confocal
> > and multiphoton imaging and had already decided for the FV1000
> > when
> > Zeiss released the LSM780.
> > After testing the LSM780 we changed our mind and decided for Zeiss.
> > For our purposes the LSM780 has exactly the improvements over
> > the 710,
> > we were looking for and therefore beat the Olympus.
> >
> > Best,
> > Stephan
> >
> >
> > ----- Original Message -----
> > From: Vladimir Gukassyan <[hidden email]>
> > Date: Tuesday, March 9, 2010 1:18 pm
> > Subject: Zeiss or Olympus
> > To: [hidden email]
> >
> > > Dear List Members,
> > >
> > > We're making a selection between Zeiss 710 and Olympus FV1000
> > for the
> > > multiphoton imaging.
> > > I would be thankful for all user experience cases and
> > suggestions on
> > > this matter.
> > >
> > > Thank you in advance.
> > > With regards,
> > > Vladimir
> > >
> > > ---------------------------------
> > > Vladimir Ghukasyan
> > > Confocal and Multiphoton Imaging Core,
> > > Neuroscience Research Center
> > > University of North Carolina
> > > 115 Mason Farm Rd., Bld. 245, 7 Fl.
> > > Chapel Hill, NC 27599-7250
> > > Tel.: [phone]
> > >
> >
> >
> > --
> > BEGIN-ANTISPAM-VOTING-LINKS
> > ------------------------------------------------------
> >
> > Teach CanIt if this mail (ID 1010315472) is spam:
> > Spam:      
> > https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=sNot 
> spam:    https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=n
> > Forget vote:
> > https://antispam.osu.edu/b.php?i=1010315472&m=06308eddccc4&c=f---
> > ---------------------------------------------------
> > END-ANTISPAM-VOTING-LINKS
> >

[RICHARD BURRY]

Mark
 
I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
Dick Burry

----- Original Message -----
From: Mark Cannell <[hidden email]>
Date: Tuesday, March 9, 2010 6:33 pm
Subject: Re: Zeiss or Olympus
To: [hidden email]

> Hi RICHARD
>
> As far as I know, for PMT based systems the dynamic range does
> not
> depend on PMT sensitivity (which is determined by the
> photocathode) but
> on gain (=PMT HV) and A/D settings.
>
> Cheers Mark
>
> BURRY wrote:
> >
> > Vladimir and Stephan
> >
> > 
> >
> > The Zeiss 780 is an upgrade for the single photon.  The
> big issues for
> > me with the Zeiss and Olympus multiphoton are the detector
> dynamic
> > range, and detector sensitivity.  Extremely sensitive
> detectors can
> > have a limited dynamic range.  I would be interested in
> hearing from
> > someone who had recently tested not only the sensitivity of
> these two
> > multiphotons, but also the dynamic range in an image collected
> a
> > moderate to low fluorescence levels.  My evaluation a
> year ago was
> > that the Olympus multiphoton had the better combination of
> sensitivity
> > and dynamic range.
> >
> > 
> >
> > Dick Burry
> >
> >
> > ----- Original Message -----
> > From: Stephan Speier <[hidden email]>
> > Date: Tuesday, March 9, 2010 8:10 am
> > Subject: Re: Zeiss or Olympus
> > To: [hidden email]
> >
> > > Dear Vladimir,
> > >
> > > We tested the Zeiss LSM710 and the Olympus FV1000 regarding
> > > confocal
> > > and multiphoton imaging and had already decided for the FV1000
> > > when
> > > Zeiss released the LSM780.
> > > After testing the LSM780 we changed our mind and decided for
> Zeiss.> > For our purposes the LSM780 has exactly the
> improvements over
> > > the 710,
> > > we were looking for and therefore beat the Olympus.
> > >
> > > Best,
> > > Stephan
> > >
> > >
> > > ----- Original Message -----
> > > From: Vladimir Gukassyan <[hidden email]>
> > > Date: Tuesday, March 9, 2010 1:18 pm
> > > Subject: Zeiss or Olympus
> > > To: [hidden email]
> > >
> > > > Dear List Members,
> > > >
> > > > We're making a selection between Zeiss 710 and Olympus FV1000
> > > for the
> > > > multiphoton imaging.
> > > > I would be thankful for all user experience cases and
> > > suggestions on
> > > > this matter.
> > > >
> > > > Thank you in advance.
> > > > With regards,
> > > > Vladimir
> > > >
> > > > ---------------------------------
> > > > Vladimir Ghukasyan
> > > > Confocal and Multiphoton Imaging Core,
> > > > Neuroscience Research Center
> > > > University of North Carolina
> > > > 115 Mason Farm Rd., Bld. 245, 7 Fl.
> > > > Chapel Hill, NC 27599-7250
> > > > Tel.: [phone]
> > > >
> > >
> > >
> > > --
> > >
>
>
> --
> BEGIN-ANTISPAM-VOTING-LINKS
> ------------------------------------------------------
>
> Teach CanIt if this mail (ID 1010662020) is spam:
> Spam:       
> https://antispam.osu.edu/b.php?i=1010662020&m=f29e0aefb64a&c=sNot spam:    https://antispam.osu.edu/b.php?i=1010662020&m=f29e0aefb64a&c=n
> Forget vote:
> https://antispam.osu.edu/b.php?i=1010662020&m=f29e0aefb64a&c=f---
> ---------------------------------------------------
> END-ANTISPAM-VOTING-LINKS
>

Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

[Axel Kurt Preuss]

Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to  

  balance the  gain   versus noise.  )

Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT   

 

 

 

 

Thanks

 

Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of RICHARD BURRY
Sent: Wednesday, March 10, 2010 8:17 AM
To: [hidden email]
Subject: Re: Zeiss or Olympus

 

Mark
 
I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
Dick Burry

----- Original Message -----
From: Mark Cannell <[hidden email]>
Date: Tuesday, March 9, 2010 6:33 pm
Subject: Re: Zeiss or Olympus
To: [hidden email]

> Hi RICHARD
>
> As far as I know, for PMT based systems the dynamic range does
> not
> depend on PMT sensitivity (which is determined by the
> photocathode) but
> on gain (=PMT HV) and A/D settings.
>
> Cheers Mark
>
> BURRY wrote:
> >
> > Vladimir and Stephan
> >
> > 
> >
> > The Zeiss 780 is an upgrade for the single photon.  The
> big issues for
> > me with the Zeiss and Olympus multiphoton are the detector
> dynamic
> > range, and detector sensitivity.  Extremely sensitive
> detectors can
> > have a limited dynamic range.  I would be interested in
> hearing from
> > someone who had recently tested not only the sensitivity of
> these two
> > multiphotons, but also the dynamic range in an image collected
> a
> > moderate to low fluorescence levels.  My evaluation a
> year ago was
> > that the Olympus multiphoton had the better combination of
> sensitivity
> > and dynamic range.
> >
> > 
> >
> > Dick Burry
> >
> >
> > ----- Original Message -----
> > From: Stephan Speier <[hidden email]>
> > Date: Tuesday, March 9, 2010 8:10 am
> > Subject: Re: Zeiss or Olympus
> > To: [hidden email]
> >
> > > Dear Vladimir,
> > >
> > > We tested the Zeiss LSM710 and the Olympus FV1000 regarding
> > > confocal
> > > and multiphoton imaging and had already decided for the FV1000
> > > when
> > > Zeiss released the LSM780.
> > > After testing the LSM780 we changed our mind and decided for
> Zeiss.> > For our purposes the LSM780 has exactly the
> improvements over
> > > the 710,
> > > we were looking for and therefore beat the Olympus.
> > >
> > > Best,
> > > Stephan
> > >
> > >
> > > ----- Original Message -----
> > > From: Vladimir Gukassyan <[hidden email]>
> > > Date: Tuesday, March 9, 2010 1:18 pm
> > > Subject: Zeiss or Olympus
> > > To: [hidden email]
> > >
> > > > Dear List Members,
> > > >
> > > > We're making a selection between Zeiss 710 and Olympus FV1000
> > > for the
> > > > multiphoton imaging.
> > > > I would be thankful for all user experience cases and
> > > suggestions on
> > > > this matter.
> > > >
> > > > Thank you in advance.
> > > > With regards,
> > > > Vladimir
> > > >
> > > > ---------------------------------
> > > > Vladimir Ghukasyan
> > > > Confocal and Multiphoton Imaging Core,
> > > > Neuroscience Research Center
> > > > University of North Carolina
> > > > 115 Mason Farm Rd., Bld. 245, 7 Fl.
> > > > Chapel Hill, NC 27599-7250
> > > > Tel.: [phone]
> > > >
> > >
> > >
> > > --
> > >
>
>
> --
> BEGIN-ANTISPAM-VOTING-LINKS
> ------------------------------------------------------
>
> Teach CanIt if this mail (ID 1010662020) is spam:
> Spam:       
> https://antispam.osu.edu/b.php?i=1010662020&m=f29e0aefb64a&c=sNot spam:    https://antispam.osu.edu/b.php?i=1010662020&m=f29e0aefb64a&c=n
> Forget vote:
> https://antispam.osu.edu/b.php?i=1010662020&m=f29e0aefb64a&c=f---
> ---------------------------------------------------
> END-ANTISPAM-VOTING-LINKS
>

Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

[Andreas Bruckbauer]

Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate) detector, not GaAs, the difference in quantum efficiency can be seen e.g. in the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The drawback is that GaAsP QE dropps dramatically for wavelength > 700 nm, but they put a normal PMTs as the two additional channels on the 780, to cover the extended range. By the way GaAsP detectors are PMTs as well, it is just a different material of the photocathode, afterwards the photoelectrons are multiplied in the same way. GaAsP detectors reach 40% quantum efficiency which is about twice as sensitive as a normal PMT. APDs have 60-70% and a back-thinned CCD about 90%., so still a lot of signal is thrown away, not to mention the losses on the way to the detector.

Andreas

-----Original Message-----
From: Axel Kurt Preuss <[hidden email]>
To: [hidden email]
Sent: Wed, 10 Mar 2010 3:08
Subject: Re: Zeiss or Olympus

Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to  

  balance the  gain   versus noise.  )

Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT   

 

 

 

 

Thanks

 

Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622

 

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of RICHARD BURRY
Sent: Wednesday, March 10, 2010 8:17 AM
To: [hidden email]
Subject: Re: Zeiss or Olympus

 

Mark
 
I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
Dick Burry

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

[Steffen Dietzel]

While we are on the GaAsPs: Is it true that they
have a half-life of only a year? I have heard
that they decay pretty fast, independant of
usage, meaning that they loose sensitivity over
time. But I didn't have a chance to ask somebody
who has acutally worked with them.

Steffen

At 09:52 10.03.2010, you wrote:

>Just to clarify, the 780 has a GaAsP (Gallium
>Arsenite Phosphate) detector, not GaAs, the
>difference in quantum efficiency can be seen
>e.g. in the Webb multiphoton review (Nature
>Biotechnology 2003, 21, 1369). The drawback is
>that GaAsP QE dropps dramatically for
>wavelength > 700 nm, but they put a normal PMTs
>as the two additional channels on the 780, to
>cover the extended range. By the way GaAsP
>detectors are PMTs as well, it is just a
>different material of the photocathode,
>afterwards the photoelectrons are multiplied in
>the same way. GaAsP detectors reach 40% quantum
>efficiency which is about twice as sensitive as
>a normal PMT. APDs have 60-70% and a
>back-thinned CCD about 90%., so still a lot of
>signal is thrown away, not to mention the losses on the way to the detector.
>
>Andreas

--
---------------------------------------------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)

[Vladimir Ghukasyan-2]

Dear Steffen,

We were operating our H7422-P40 (Hamamatsu) for 4 years and I didn't
notice a significant decay with it.

Regards,
Vladimir


On Wed, Mar 10, 2010 at 8:06 AM, Steffen Dietzel <[hidden email]> wrote:

> While we are on the GaAsPs: Is it true that they have a half-life of only a
> year? I have heard that they decay pretty fast, independant of usage,
> meaning that they loose sensitivity over time. But I didn't have a chance to
> ask somebody who has acutally worked with them.
>
> Steffen
>
> At 09:52 10.03.2010, you wrote:
>>
>> Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate)
>> detector, not GaAs, the difference in quantum efficiency can be seen e.g. in
>> the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The
>> drawback is that GaAsP QE dropps dramatically for wavelength > 700 nm, but
>> they put a normal PMTs as the two additional channels on the 780, to cover
>> the extended range. By the way GaAsP detectors are PMTs as well, it is just
>> a different material of the photocathode, afterwards the photoelectrons are
>> multiplied in the same way. GaAsP detectors reach 40% quantum efficiency
>> which is about twice as sensitive as a normal PMT. APDs have 60-70% and a
>> back-thinned CCD about 90%., so still a lot of signal is thrown away, not to
>> mention the losses on the way to the detector.
>>
>> Andreas
>
>
> --
> ---------------------------------------------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>

[James Pawley]

Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate) detector, not GaAs, the difference in quantum efficiency can be seen e.g. in the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The drawback is that GaAsP QE drops dramatically for wavelength > 700 nm, but they put a normal PMTs as the two additional channels on the 780, to cover the extended range. By the way GaAsP detectors are PMTs as well, it is just a different material of the photocathode, afterwards the photoelectrons are multiplied in the same way. GaAsP detectors reach 40% quantum efficiency which is about twice as sensitive as a normal PMT. APDs have 60-70% and a back-thinned CCD about 90%., so still a lot of signal is thrown away, not to mention the losses on the way to the detector.

Andreas

Hi all,

Indeed, the GaAs and GaAs phosphide QE curves are very impressive. However, it is important to remember what is actually measured to make these curves. PMT curves refer to the fraction of photons striking the photocathode that produce photoelectrons (It is usually measured by allowing a calibrated amount of light to strike the photocathode and using a nano-ammeter to sense the total photoelectron current between the photocathode and all the other electrodes in the PMT). However, depending on the electrode geometry, 10-30% of these photoelectrons don't actually hit the first dynode (D1), and therefore do not contribute to the PMT output.

Of those photoelectrons that do hit D1, a reasonable fraction fail to excite any secondary electrons, and again, do not contribute to the PMT output. There are many reasons for this but one is just Poisson statistics. If the average gain is say 4, then about 8% of the collisions will result in zero electrons being emitted. However, this effect is again multiplied by geometrical factors where SE produced in different parts of D1 have better or worse chances of actually striking D2 and producing a SE. Signal loss in this way depends a lot on the actual voltage between the photocathode and D1: it will be less when the voltage is higher. Unfortunately, few confocals seem to have been set up in such  way that this is always true. On average signal loss by failure to propagate after collision with D1 will be an additional 20-40%.

Finally, the same type of Poisson effects that cause some signal to be lost entirely, cause the amount by which the remainder is amplified to be highly variable (10-90%). This variation degrades the accuracy of the output signal by introducing what is called multiplicative noise. Because this extra noise can only be "overcome" by counting more photons, its presence effectively reduces the effective QE of the device. In the best case, this reduction is about 40% and in the worst case (an exponential gain distribution, approximated by some micro PMTs) 75% (i.e., the QE is reduced to 60% or 25% of what it would have been if all photoelectrons were equally amplified).

As a result, while the peak effective QE of a PMT with a GaAs or GaAsP photocathode is indeed much better than that of the more common S-20 photocathode, in terms of its effectiveness in providing an output current that is proportional to the input photon signal, the QE is more in the range of 3 -10% (depending on dynode geometry and first-dynode voltage) than 40%. (The 60% numbers are for APDs rather than for a GaAs or GaAsP photocathode on a PMT.)

The performance can be improved somewhat on the few confocals that allow single-photon counting as this procedure eliminates multiplicative noise. (see below about the limitations imposed by photon counting)

This tedious recital is I hope  justified by noting that, at least when EG&G was the major APD supplier, APD performance was not specified in terms of QE but as Photon Detection Efficiency (PDE). Although APDs can be operated in a low gain, proportional mode, their PDE under these conditions is very low (because APD multiplicative noise is very high and at low (non-avalanche breakdown) gain, by far the most likely gain of the initial photoelectron is zero).

Therefore, high PDE (or high QE) AOD units tend to operate at high bias (high, avalanche gain) and this requires circuitry to quench the avalanche breakdown and count the single-photon pulses. Modern units contain both the sensor itself and the pulse counting and avalanche quenching circuits needed for counting the single-photon pulses. In other words (assuming that Hamamatsu follows the EG&G precedent), their QE figures for single-photon counting units already include any losses for non-propagation or multiplicative noise. Therefore, a quoted PID of 60% really does mean that 60% of the photons (of the specified wavelength) that strike the center of the active surface will be accurately counted.

This is about 4-10x better than the performance of a similar GaAs or GaAsP photocathode on a PMT.

This good news is tempered by the fact that, because of the high capacitance of the AOD itself, it is hard to count much faster than, say 30MHz. As 30MHz comes out to an absolute maximum of 60 counts during a 2 µs pixel, this means that at least 50% of your counts will be lost due to pulse pileup when 30 counts arrive per pixel and 10% will be lost at only 6 counts/pixel. In other words one has to be very careful to adjust the excitation intensity so as not to "clip" the brightness of those parts of the image that contain a lot of fluorophor.

Lots more on this in The Handbook,

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.

Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to  

  balance the  gain   versus noise.  )

Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT   

 

 

 

 

Thanks

 

Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622

 

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of RICHARD BURRY
Sent: Wednesday, March 10, 2010 8:17 AM
To: [hidden email]
Subject: Re: Zeiss or Olympus

 

Mark
 
I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
Dick Burry

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

--

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

[RICHARD BURRY]

Jim
 
Thanks for the details of the GaAs and GaAsP detectors.  But my question remains, does Zeiss use either of these detectors in its 780 multiphoton?  And does this represent an improvement in S/N over standard PMTs?  My experience is with the NLO a while ago is that the GaAs detector was more sensitive but for samples with a range of emission intensity, it was difficult to not saturate some part of the sample.
 
Dick

----- Original Message -----
From: James Pawley <[hidden email]>
Date: Wednesday, March 10, 2010 11:58 am
Subject: Re: Zeiss or Olympus
To: [hidden email]

> Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate) detector, not GaAs, the difference in quantum efficiency can be seen e.g. in the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The drawback is that GaAsP QE drops dramatically for wavelength > 700 nm, but they put a normal PMTs as the two additional channels on the 780, to cover the extended range. By the way GaAsP detectors are PMTs as well, it is just a different material of the photocathode, afterwards the photoelectrons are multiplied in the same way. GaAsP detectors reach 40% quantum efficiency which is about twice as sensitive as a normal PMT. APDs have 60-70% and a back-thinned CCD about 90%., so still a lot of signal is thrown away, not to mention the losses on the way to the detector.

> Andreas

> Hi all,


> Indeed, the GaAs and GaAs phosphide QE curves are very impressive. However, it is important to remember what is actually measured to make these curves. PMT curves refer to the fraction of photons striking the photocathode that produce photoelectrons (It is usually measured by allowing a calibrated amount of light to strike the photocathode and using a nano-ammeter to sense the total photoelectron current between the photocathode and all the other electrodes in the PMT). However, depending on the electrode geometry, 10-30% of these photoelectrons don't actually hit the first dynode (D1), and therefore do not contribute to the PMT output.


> Of those photoelectrons that do hit D1, a reasonable fraction fail to excite any secondary electrons, and again, do not contribute to the PMT output. There are many reasons for this but one is just Poisson statistics. If the average gain is say 4, then about 8% of the collisions will result in zero electrons being emitted. However, this effect is again multiplied by geometrical factors where SE produced in different parts of D1 have better or worse chances of actually striking D2 and producing a SE. Signal loss in this way depends a lot on the actual voltage between the photocathode and D1: it will be less when the voltage is higher. Unfortunately, few confocals seem to have been set up in such  way that this is always true. On average signal loss by failure to propagate after collision with D1 will be an additional 20-40%.


> Finally, the same type of Poisson effects that cause some signal to be lost entirely, cause the amount by which the remainder is amplified to be highly variable (10-90%). This variation degrades the accuracy of the output signal by introducing what is called multiplicative noise. Because this extra noise can only be "overcome" by counting more photons, its presence effectively reduces the effective QE of the device. In the best case, this reduction is about 40% and in the worst case (an exponential gain distribution, approximated by some micro PMTs) 75% (i.e., the QE is reduced to 60% or 25% of what it would have been if all photoelectrons were equally amplified).


> As a result, while the peak effective QE of a PMT with a GaAs or GaAsP photocathode is indeed much better than that of the more common S-20 photocathode, in terms of its effectiveness in providing an output current that is proportional to the input photon signal, the QE is more in the range of 3 -10% (depending on dynode geometry and first-dynode voltage) than 40%. (The 60% numbers are for APDs rather than for a GaAs or GaAsP photocathode on a PMT.)


> The performance can be improved somewhat on the few confocals that allow single-photon counting as this procedure eliminates multiplicative noise. (see below about the limitations imposed by photon counting)


> This tedious recital is I hope  justified by noting that, at least when EG&G was the major APD supplier, APD performance was not specified in terms of QE but as Photon Detection Efficiency (PDE). Although APDs can be operated in a low gain, proportional mode, their PDE under these conditions is very low (because APD multiplicative noise is very high and at low (non-avalanche breakdown) gain, by far the most likely gain of the initial photoelectron is zero).


> Therefore, high PDE (or high QE) AOD units tend to operate at high bias (high, avalanche gain) and this requires circuitry to quench the avalanche breakdown and count the single-photon pulses. Modern units contain both the sensor itself and the pulse counting and avalanche quenching circuits needed for counting the single-photon pulses. In other words (assuming that Hamamatsu follows the EG&G precedent), their QE figures for single-photon counting units already include any losses for non-propagation or multiplicative noise. Therefore, a quoted PID of 60% really does mean that 60% of the photons (of the specified wavelength) that strike the center of the active surface will be accurately counted.


> This is about 4-10x better than the performance of a similar GaAs or GaAsP photocathode on a PMT.


> This good news is tempered by the fact that, because of the high capacitance of the AOD itself, it is hard to count much faster than, say 30MHz. As 30MHz comes out to an absolute maximum of 60 counts during a 2 µs pixel, this means that at least 50% of your counts will be lost due to pulse pileup when 30 counts arrive per pixel and 10% will be lost at only 6 counts/pixel. In other words one has to be very careful to adjust the excitation intensity so as not to "clip" the brightness of those parts of the image that contain a lot of fluorophor.


> Lots more on this in The Handbook,


> 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.

> Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to  

  balance the  gain   versus noise.  )

Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT   

 

 

 

 

Thanks

 

Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622

 

---

From: Confocal Microscopy List [<A href="javascript:main.compose('new', 't=[e-mail]&amp;')">mailto:CONFOCALMICROSCOPY@...] On Behalf Of RICHARD BURRY
> Sent: Wednesday, March 10, 2010 8:17 AM
> To: <A href="javascript:main.compose('new', 't=[e-mail]')">CONFOCALMICROSCOPY@...
> Subject: Re: Zeiss or Olympus

 

Mark
 
> I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
> Dick Burry

> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

> --  > 

              **********************************************
> 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.

---

> Spam
> Not spam
> Forget previous vote

Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

[Guy Cox-2]

The other thing about APDs is that they are so vulnerable to excessive light that one bright pixel will trigger the safety circuit and the rest of your image is blank (QE = 0)!

 

                                                                Guy

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Thursday, 11 March 2010 3:58 AM
To: [hidden email]
Subject: Re: Zeiss or Olympus

 

Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate) detector, not GaAs, the difference in quantum efficiency can be seen e.g. in the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The drawback is that GaAsP QE drops dramatically for wavelength > 700 nm, but they put a normal PMTs as the two additional channels on the 780, to cover the extended range. By the way GaAsP detectors are PMTs as well, it is just a different material of the photocathode, afterwards the photoelectrons are multiplied in the same way. GaAsP detectors reach 40% quantum efficiency which is about twice as sensitive as a normal PMT. APDs have 60-70% and a back-thinned CCD about 90%., so still a lot of signal is thrown away, not to mention the losses on the way to the detector.

 

Andreas

Hi all,

 

Indeed, the GaAs and GaAs phosphide QE curves are very impressive. However, it is important to remember what is actually measured to make these curves. PMT curves refer to the fraction of photons striking the photocathode that produce photoelectrons (It is usually measured by allowing a calibrated amount of light to strike the photocathode and using a nano-ammeter to sense the total photoelectron current between the photocathode and all the other electrodes in the PMT). However, depending on the electrode geometry, 10-30% of these photoelectrons don't actually hit the first dynode (D1), and therefore do not contribute to the PMT output.

 

Of those photoelectrons that do hit D1, a reasonable fraction fail to excite any secondary electrons, and again, do not contribute to the PMT output. There are many reasons for this but one is just Poisson statistics. If the average gain is say 4, then about 8% of the collisions will result in zero electrons being emitted. However, this effect is again multiplied by geometrical factors where SE produced in different parts of D1 have better or worse chances of actually striking D2 and producing a SE. Signal loss in this way depends a lot on the actual voltage between the photocathode and D1: it will be less when the voltage is higher. Unfortunately, few confocals seem to have been set up in such  way that this is always true. On average signal loss by failure to propagate after collision with D1 will be an additional 20-40%.

 

Finally, the same type of Poisson effects that cause some signal to be lost entirely, cause the amount by which the remainder is amplified to be highly variable (10-90%). This variation degrades the accuracy of the output signal by introducing what is called multiplicative noise. Because this extra noise can only be "overcome" by counting more photons, its presence effectively reduces the effective QE of the device. In the best case, this reduction is about 40% and in the worst case (an exponential gain distribution, approximated by some micro PMTs) 75% (i.e., the QE is reduced to 60% or 25% of what it would have been if all photoelectrons were equally amplified).

 

As a result, while the peak effective QE of a PMT with a GaAs or GaAsP photocathode is indeed much better than that of the more common S-20 photocathode, in terms of its effectiveness in providing an output current that is proportional to the input photon signal, the QE is more in the range of 3 -10% (depending on dynode geometry and first-dynode voltage) than 40%. (The 60% numbers are for APDs rather than for a GaAs or GaAsP photocathode on a PMT.)

 

The performance can be improved somewhat on the few confocals that allow single-photon counting as this procedure eliminates multiplicative noise. (see below about the limitations imposed by photon counting)

 

This tedious recital is I hope  justified by noting that, at least when EG&G was the major APD supplier, APD performance was not specified in terms of QE but as Photon Detection Efficiency (PDE). Although APDs can be operated in a low gain, proportional mode, their PDE under these conditions is very low (because APD multiplicative noise is very high and at low (non-avalanche breakdown) gain, by far the most likely gain of the initial photoelectron is zero).

 

Therefore, high PDE (or high QE) AOD units tend to operate at high bias (high, avalanche gain) and this requires circuitry to quench the avalanche breakdown and count the single-photon pulses. Modern units contain both the sensor itself and the pulse counting and avalanche quenching circuits needed for counting the single-photon pulses. In other words (assuming that Hamamatsu follows the EG&G precedent), their QE figures for single-photon counting units already include any losses for non-propagation or multiplicative noise. Therefore, a quoted PID of 60% really does mean that 60% of the photons (of the specified wavelength) that strike the center of the active surface will be accurately counted.

 

This is about 4-10x better than the performance of a similar GaAs or GaAsP photocathode on a PMT.

 

This good news is tempered by the fact that, because of the high capacitance of the AOD itself, it is hard to count much faster than, say 30MHz. As 30MHz comes out to an absolute maximum of 60 counts during a 2 µs pixel, this means that at least 50% of your counts will be lost due to pulse pileup when 30 counts arrive per pixel and 10% will be lost at only 6 counts/pixel. In other words one has to be very careful to adjust the excitation intensity so as not to "clip" the brightness of those parts of the image that contain a lot of fluorophor.

 

Lots more on this in The Handbook,

 

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.

 

 

Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to  

  balance the  gain   versus noise.  )

Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT   

 

 

 

 

Thanks

 

Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622

 

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of RICHARD BURRY
Sent: Wednesday, March 10, 2010 8:17 AM
To: [hidden email]
Subject: Re: Zeiss or Olympus

 

Mark
 
I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
Dick Burry

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

 

 

-- 

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

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.733 / Virus Database: 271.1.1/2710 - Release Date: 03/10/10 06:33:00

[Andreas Bruckbauer]

From the Zeiss Brochure:

The new GaAsP detector technology is not confined to visual
light excitation. The LSM BiG (binary GaAsP) now also offers
you multicolor multiphoton imaging with GaAsP performance.
Two LSM BiG modules can be added to NLO systems
as transmitted and incident light NDDs, providing 4 ultrasensitive
detection channels. The LSM 780 NLO and LSM 710 NLO
let you penetrate deeper and detect more light.

So additional to the internal GaAsP spectral detector you can add a GaAsP NDD (BIG detector) for multiphoton imaging. And you can have the GaAsP single channel sitting directly over the objective. I think you can also couple two GaAsP detectors on an external port of the scan head

best wishes

Andreas

-----Original Message-----
From: RICHARD BURRY <[hidden email]>
To: [hidden email]
Sent: Wed, 10 Mar 2010 18:54
Subject: Re: Zeiss or Olympus

Jim
 
Thanks for the details of the GaAs and GaAsP detectors.  But my question remains, does Zeiss use either of these detectors in its 780 multiphoton?  And does this represent an improvement in S/N over standard PMTs?  My experience is with the NLO a while ago is that the GaAs detector was more sensitive but for samples with a range of emission intensity, it was difficult to not saturate some part of the sample.
 
Dick

----- Original Message -----
From: James Pawley <[hidden email]>
Date: Wednesday, March 10, 2010 11:58 am
Subject: Re: Zeiss or Olympus
To: [hidden email]

> Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate) detector, not GaAs, the difference in quantum efficiency can be seen e.g. in the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The drawback is that GaAsP QE drops dramatically for wavelength > 700 nm, but they put a normal PMTs as the two additional channels on the 780, to cover the extended range. By the way GaAsP detectors are PMTs as well, it is just a different material of the photocathode, afterwards the photoelectrons are multiplied in the same way. GaAsP detectors reach 40% quantum efficiency which is about twice as sensitive as a normal PMT. APDs have 60-70% and a back-thinned CCD about 90%., so still a lot of signal is thrown away, not to mention the losses on the way to the detector.

> Andreas

> Hi all,


> Indeed, the GaAs and GaAs phosphide QE curves are very impressive. However, it is important to remember what is actually measured to make these curves. PMT curves refer to the fraction of photons striking the photocathode that produce photoelectrons (It is usually measured by allowing a calibrated amount of light to strike the photocathode and using a nano-ammeter to sense the total photoelectron current between the photocathode and all the other electrodes in the PMT). However, depending on the electrode geometry, 10-30% of these photoelectrons don't actually hit the first dynode (D1), and therefore do not contribute to the PMT output.


> Of those photoelectrons that do hit D1, a reasonable fraction fail to excite any secondary electrons, and again, do not contribute to the PMT output. There are many reasons for this but one is just Poisson statistics. If the average gain is say 4, then about 8% of the collisions will result in zero electrons being emitted. However, this effect is again multiplied by geometrical factors where SE produced in different parts of D1 have better or worse chances of actually striking D2 and producing a SE. Signal loss in this way depends a lot on the actual voltage between the photocathode and D1: it will be less when the voltage is higher. Unfortunately, few confocals seem to have been set up in such  way that this is always true. On average signal loss by failure to propagate after collision with D1 will be an additional 20-40%.


> Finally, the same type of Poisson effects that cause some signal to be lost entirely, cause the amount by which the remainder is amplified to be highly variable (10-90%). This variation degrades the accuracy of the output signal by introducing what is called multiplicative noise. Because this extra noise can only be "overcome" by counting more photons, its presence effectively reduces the effective QE of the device. In the best case, this reduction is about 40% and in the worst case (an exponential gain distribution, approximated by some micro PMTs) 75% (i.e., the QE is reduced to 60% or 25% of what it would have been if all photoelectrons were equally amplified).


> As a result, while the peak effective QE of a PMT with a GaAs or GaAsP photocathode is indeed much better than that of the more common S-20 photocathode, in terms of its effectiveness in providing an output current that is proportional to the input photon signal, the QE is more in the range of 3 -10% (depending on dynode geometry and first-dynode voltage) than 40%. (The 60% numbers are for APDs rather than for a GaAs or GaAsP photocathode on a PMT.)


> The performance can be improved somewhat on the few confocals that allow single-photon counting as this procedure eliminates multiplicative noise. (see below about the limitations imposed by photon counting)


> This tedious recital is I hope  justified by noting that, at least when EG&G was the major APD supplier, APD performance was not specified in terms of QE but as Photon Detection Efficiency (PDE). Although APDs can be operated in a low gain, proportional mode, their PDE under these conditions is very low (because APD multiplicative noise is very high and at low (non-avalanche breakdown) gain, by far the most likely gain of the initial photoelectron is zero).


> Therefore, high PDE (or high QE) AOD units tend to operate at high bias (high, avalanche gain) and this requires circuitry to quench the avalanche breakdown and count the single-photon pulses. Modern units contain both the sensor itself and the pulse counting and avalanche quenching circuits needed for counting the single-photon pulses. In other words (assuming that Hamamatsu follows the EG&G precedent), their QE figures for single-photon counting units already include any losses for non-propagation or multiplicative noise. Therefore, a quoted PID of 60% really does mean that 60% of the photons (of the specified wavelength) that strike the center of the active surface will be accurately counted.


> This is about 4-10x better than the performance of a similar GaAs or GaAsP photocathode on a PMT.


> This good news is tempered by the fact that, because of the high capacitance of the AOD itself, it is hard to count much faster than, say 30MHz. As 30MHz comes out to an absolute maximum of 60 counts during a 2 µs pixel, this means that at least 50% of your counts will be lost due to pulse pileup when 30 counts arrive per pixel and 10% will be lost at only 6 counts/pixel. In other words one has to be very careful to adjust the excitation intensity so as not to "clip" the brightness of those parts of the image that contain a lot of fluorophor.


> Lots more on this in The Handbook,


> 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.

> Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to  

  balance the  gain   versus noise.  )

Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT   

 

 

 

 

Thanks

 

Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of RICHARD BURRY
> Sent: Wednesday, March 10, 2010 8:17 AM
> To: [hidden email]
> Subject: Re: Zeiss or Olympus

 

Mark
 
> I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
> Dick Burry

> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

> --  > 

              **********************************************
> 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.

---

> Spam
> Not spam
> Forget previous vote

Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

[Andreas Bruckbauer]

Does anyone  know  a source  for a 100 mW 491 nm laser module which can be easily modulated  by an external voltage?
Thanks, Andreas

[James Pawley]

From the Zeiss Brochure:

The new GaAsP detector technology is not confined to visual
light excitation. The LSM BiG (binary GaAsP) now also offers
you multicolor multiphoton imaging with GaAsP performance.
Two LSM BiG modules can be added to NLO systems
as transmitted and incident light NDDs, providing 4 ultrasensitive
detection channels. The LSM 780 NLO and LSM 710 NLO
let you penetrate deeper and detect more light.

So additional to the internal GaAsP spectral detector you can add a GaAsP NDD (BIG detector) for multiphoton imaging. And you can have the GaAsP single channel sitting directly over the objective. I think you can also couple two GaAsP detectors on an external port of the scan head

best wishes

Andreas

Thanks Andreas,

Does anyone have any idea of the maker or model number of these BiG detectors?

As I mentioned earlier, the count-rate performance of APD single-photon counting systems are limited by the capacitance of the sensor diode. This is in turn proportional to the the sensitive area of the detector.

For this reason, the sensitive areas tend to be small: maybe 100µm diameter. As this is much smaller than the ray bundle coming out the back of most high-mag, high-NA objectives, it is not obvious how one could "squeeze" the light bundle there to match the sensitive area. So I assume that the word "big" in your post is relevant and it would be nice to know how big and if it works in the counting mode, and whether it is an avalanche diode or merely a GaAsP photodiode (no amplification, but no multiplicative noise either. Very suitable for transmitted detectors....).

Cheers,

Jim P.

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

-----Original Message-----
From: RICHARD BURRY <[hidden email]>
To: [hidden email]
Sent: Wed, 10 Mar 2010 18:54
Subject: Re: Zeiss or Olympus

Jim
 
Thanks for the details of the GaAs and GaAsP detectors.  But my question remains, does Zeiss use either of these detectors in its 780 multiphoton?  And does this represent an improvement in S/N over standard PMTs?  My experience is with the NLO a while ago is that the GaAs detector was more sensitive but for samples with a range of emission intensity, it was difficult to not saturate some part of the sample.
 
Dick

----- Original Message -----
From: James Pawley <[hidden email]>
Date: Wednesday, March 10, 2010 11:58 am
Subject: Re: Zeiss or Olympus
To: [hidden email]

> Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate) detector, not GaAs, the difference in quantum efficiency can be seen e.g. in the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The drawback is that GaAsP QE drops dramatically for wavelength > 700 nm, but they put a normal PMTs as the two additional channels on the 780, to cover the extended range. By the way GaAsP detectors are PMTs as well, it is just a different material of the photocathode, afterwards the photoelectrons are multiplied in the same way. GaAsP detectors reach 40% quantum efficiency which is about twice as sensitive as a normal PMT. APDs have 60-70% and a back-thinned CCD about 90%., so still a lot of signal is thrown away, not to mention the losses on the way to the detector.

> Andreas

> Hi all,


> Indeed, the GaAs and GaAs phosphide QE curves are very impressive. However, it is important to remember what is actually measured to make these curves. PMT curves refer to the fraction of photons striking the photocathode that produce photoelectrons (It is usually measured by allowing a calibrated amount of light to strike the photocathode and using a nano-ammeter to sense the total photoelectron current between the photocathode and all the other electrodes in the PMT). However, depending on the electrode geometry, 10-30% of these photoelectrons don't actually hit the first dynode (D1), and therefore do not contribute to the PMT output.

> Of those photoelectrons that do hit D1, a reasonable fraction fail to excite any secondary electrons, and again, do not contribute to the PMT output. There are many reasons for this but one is just Poisson statistics. If the average gain is say 4, then about 8% of the collisions will result in zero electrons being emitted. However, this effect is again multiplied by geometrical factors where SE produced in different parts of D1 have better or worse chances of actually striking D2 and producing a SE. Signal loss in this way depends a lot on the actual voltage between the photocathode and D1: it will be less when the voltage is higher. Unfortunately, few confocals seem to have been set up in such  way that this is always true. On average signal loss by failure to propagate after collision with D1 will be an additional 20-40%.


> Finally, the same type of Poisson effects that cause some signal to be lost entirely, cause the amount by which the remainder is amplified to be highly variable (10-90%). This variation degrades the accuracy of the output signal by introducing what is called multiplicative noise. Because this extra noise can only be "overcome" by counting more photons, its presence effectively reduces the effective QE of the device. In the best case, this reduction is about 40% and in the worst case (an exponential gain distribution, approximated by some micro PMTs) 75% (i.e., the QE is reduced to 60% or 25% of what it would have been if all photoelectrons were equally amplified).


> As a result, while the peak effective QE of a PMT with a GaAs or GaAsP photocathode is indeed much better than that of the more common S-20 photocathode, in terms of its effectiveness in providing an output current that is proportional to the input photon signal, the QE is more in the range of 3 -10% (depending on dynode geometry and first-dynode voltage) than 40%. (The 60% numbers are for APDs rather than for a GaAs or GaAsP photocathode on a PMT.)


> The performance can be improved somewhat on the few confocals that allow single-photon counting as this procedure eliminates multiplicative noise. (see below about the limitations imposed by photon counting)


> This tedious recital is I hope  justified by noting that, at least when EG&G was the major APD supplier, APD performance was not specified in terms of QE but as Photon Detection Efficiency (PDE). Although APDs can be operated in a low gain, proportional mode, their PDE under these conditions is very low (because APD multiplicative noise is very high and at low (non-avalanche breakdown) gain, by far the most likely gain of the initial photoelectron is zero).


> Therefore, high PDE (or high QE) AOD units tend to operate at high bias (high, avalanche gain) and this requires circuitry to quench the avalanche breakdown and count the single-photon pulses. Modern units contain both the sensor itself and the pulse counting and avalanche quenching circuits needed for counting the single-photon pulses. In other words (assuming that Hamamatsu follows the EG&G precedent), their QE figures for single-photon counting units already include any losses for non-propagation or multiplicative noise. Therefore, a quoted PID of 60% really does mean that 60% of the photons (of the specified wavelength) that strike the center of the active surface will be accurately counted.


> This is about 4-10x better than the performance of a similar GaAs or GaAsP photocathode on a PMT.


> This good news is tempered by the fact that, because of the high capacitance of the AOD itself, it is hard to count much faster than, say 30MHz. As 30MHz comes out to an absolute maximum of 60 counts during a 2 µs pixel, this means that at least 50% of your counts will be lost due to pulse pileup when 30 counts arrive per pixel and 10% will be lost at only 6 counts/pixel. In other words one has to be very careful to adjust the excitation intensity so as not to "clip" the brightness of those parts of the image that contain a lot of fluorophor.


> Lots more on this in The Handbook,


> 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.

> Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to 

  balance the  gain   versus noise.  )

Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT  

 

 

 

 

Thanks

 

Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of RICHARD BURRY
> Sent: Wednesday, March 10, 2010 8:17 AM
> To: [hidden email]
> Subject: Re: Zeiss or Olympus

 

Mark
 
> I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties. 
 
> Dick Burry

> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

> --  >

              **********************************************
> 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.

---

> Spam
> Not spam
> Forget previous vote

Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

--

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

[Craig Brideau]

I know that the APD's that are designed as drop-in PMT replacements
use linked arrays of smaller APDs so you can have large surface area
but still keep the capacitance down.  SensL has some kind of
fast-quench circuit on their array so it quickly dumps its charge
before the next event.

Craig


On Thu, Mar 11, 2010 at 1:28 PM, James Pawley <[hidden email]> wrote:

> From the Zeiss Brochure:
>
> The new GaAsP detector technology is not confined to visual
> light excitation. The LSM BiG (binary GaAsP) now also offers
> you multicolor multiphoton imaging with GaAsP performance.
> Two LSM BiG modules can be added to NLO systems
> as transmitted and incident light NDDs, providing 4 ultrasensitive
> detection channels. The LSM 780 NLO and LSM 710 NLO
> let you penetrate deeper and detect more light.
>
> So additional to the internal GaAsP spectral detector you can add a GaAsP
> NDD (BIG detector) for multiphoton imaging. And you can have the GaAsP
> single channel sitting directly over the objective. I think you can also
> couple two GaAsP detectors on an external port of the scan head
>
> best wishes
>
> Andreas
>
> Thanks Andreas,
> Does anyone have any idea of the maker or model number of these BiG
> detectors?
> As I mentioned earlier, the count-rate performance of APD single-photon
> counting systems are limited by the capacitance of the sensor diode. This is
> in turn proportional to the the sensitive area of the detector.
> For this reason, the sensitive areas tend to be small: maybe 100µm diameter.
> As this is much smaller than the ray bundle coming out the back of most
> high-mag, high-NA objectives, it is not obvious how one could "squeeze" the
> light bundle there to match the sensitive area. So I assume that the word
> "big" in your post is relevant and it would be nice to know how big and if
> it works in the counting mode, and whether it is an avalanche diode or
> merely a GaAsP photodiode (no amplification, but no multiplicative noise
> either. Very suitable for transmitted detectors....).
> 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.
>
> -----Original Message-----
> From: RICHARD BURRY <[hidden email]>
> To: [hidden email]
> Sent: Wed, 10 Mar 2010 18:54
> Subject: Re: Zeiss or Olympus
>
> Jim
>
> Thanks for the details of the GaAs and GaAsP detectors.  But my question
> remains, does Zeiss use either of these detectors in its 780 multiphoton?
> And does this represent an improvement in S/N over standard PMTs?  My
> experience is with the NLO a while ago is that the GaAs detector was more
> sensitive but for samples with a range of emission intensity, it was
> difficult to not saturate some part of the sample.
>
> Dick
>
> ----- Original Message -----
> From: James Pawley <[hidden email]>
> Date: Wednesday, March 10, 2010 11:58 am
> Subject: Re: Zeiss or Olympus
> To: [hidden email]
>
>> Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate)
>> detector, not GaAs, the difference in quantum efficiency can be seen e.g. in
>> the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The
>> drawback is that GaAsP QE drops dramatically for wavelength > 700 nm, but
>> they put a normal PMTs as the two additional channels on the 780, to cover
>> the extended range. By the way GaAsP detectors are PMTs as well, it is just
>> a different material of the photocathode, afterwards the photoelectrons are
>> multiplied in the same way. GaAsP detectors reach 40% quantum efficiency
>> which is about twice as sensitive as a normal PMT. APDs have 60-70% and a
>> back-thinned CCD about 90%., so still a lot of signal is thrown away, not to
>> mention the losses on the way to the detector.
>
>
>> Andreas
>
>> Hi all,
>
>
>> Indeed, the GaAs and GaAs phosphide QE curves are very impressive.
>> However, it is important to remember what is actually measured to make these
>> curves. PMT curves refer to the fraction of photons striking the
>> photocathode that produce photoelectrons (It is usually measured by allowing
>> a calibrated amount of light to strike the photocathode and using a
>> nano-ammeter to sense the total photoelectron current between the
>> photocathode and all the other electrodes in the PMT). However, depending on
>> the electrode geometry, 10-30% of these photoelectrons don't actually hit
>> the first dynode (D1), and therefore do not contribute to the PMT output.
>
>> Of those photoelectrons that do hit D1, a reasonable fraction fail to
>> excite any secondary electrons, and again, do not contribute to the PMT
>> output. There are many reasons for this but one is just Poisson statistics.
>> If the average gain is say 4, then about 8% of the collisions will result in
>> zero electrons being emitted. However, this effect is again multiplied by
>> geometrical factors where SE produced in different parts of D1 have better
>> or worse chances of actually striking D2 and producing a SE. Signal loss in
>> this way depends a lot on the actual voltage between the photocathode and
>> D1: it will be less when the voltage is higher. Unfortunately, few confocals
>> seem to have been set up in such  way that this is always true. On average
>> signal loss by failure to propagate after collision with D1 will be an
>> additional 20-40%.
>
>
>> Finally, the same type of Poisson effects that cause some signal to be
>> lost entirely, cause the amount by which the remainder is amplified to be
>> highly variable (10-90%). This variation degrades the accuracy of the output
>> signal by introducing what is called multiplicative noise. Because this
>> extra noise can only be "overcome" by counting more photons, its presence
>> effectively reduces the effective QE of the device. In the best case, this
>> reduction is about 40% and in the worst case (an exponential gain
>> distribution, approximated by some micro PMTs) 75% (i.e., the QE is reduced
>> to 60% or 25% of what it would have been if all photoelectrons were equally
>> amplified).
>
>
>> As a result, while the peak effective QE of a PMT with a GaAs or GaAsP
>> photocathode is indeed much better than that of the more common S-20
>> photocathode, in terms of its effectiveness in providing an output current
>> that is proportional to the input photon signal, the QE is more in the range
>> of 3 -10% (depending on dynode geometry and first-dynode voltage) than 40%.
>> (The 60% numbers are for APDs rather than for a GaAs or GaAsP photocathode
>> on a PMT.)
>
>
>> The performance can be improved somewhat on the few confocals that allow
>> single-photon counting as this procedure eliminates multiplicative noise.
>> (see below about the limitations imposed by photon counting)
>
>
>> This tedious recital is I hope  justified by noting that, at least when
>> EG&G was the major APD supplier, APD performance was not specified in terms
>> of QE but as Photon Detection Efficiency (PDE). Although APDs can be
>> operated in a low gain, proportional mode, their PDE under these conditions
>> is very low (because APD multiplicative noise is very high and at low
>> (non-avalanche breakdown) gain, by far the most likely gain of the initial
>> photoelectron is zero).
>
>
>> Therefore, high PDE (or high QE) AOD units tend to operate at high bias
>> (high, avalanche gain) and this requires circuitry to quench the avalanche
>> breakdown and count the single-photon pulses. Modern units contain both the
>> sensor itself and the pulse counting and avalanche quenching circuits needed
>> for counting the single-photon pulses. In other words (assuming that
>> Hamamatsu follows the EG&G precedent), their QE figures for single-photon
>> counting units already include any losses for non-propagation or
>> multiplicative noise. Therefore, a quoted PID of 60% really does mean that
>> 60% of the photons (of the specified wavelength) that strike the center of
>> the active surface will be accurately counted.
>
>
>> This is about 4-10x better than the performance of a similar GaAs or GaAsP
>> photocathode on a PMT.
>
>
>> This good news is tempered by the fact that, because of the high
>> capacitance of the AOD itself, it is hard to count much faster than, say
>> 30MHz. As 30MHz comes out to an absolute maximum of 60 counts during a 2 µs
>> pixel, this means that at least 50% of your counts will be lost due to pulse
>> pileup when 30 counts arrive per pixel and 10% will be lost at only 6
>> counts/pixel. In other words one has to be very careful to adjust the
>> excitation intensity so as not to "clip" the brightness of those parts of
>> the image that contain a lot of fluorophor.
>
>
>> Lots more on this in The Handbook,
>
>
>> 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.
>
>> Just to mention, should one be stuck with PMTs instead GaAs,   one could
>> play with the applied bias voltage to modify   dark noise   (to
>
>   balance the  gain   versus noise.  )
>
> Nice thing with Olympus Kalman filtering is   that its use would allow
>   increase the bias voltage of PMT
>
>
>
>
>
>
>
>
>
> Thanks
>
>
>
> Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622
>
>
>
> ________________________________
>
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of RICHARD BURRY
>> Sent: Wednesday, March 10, 2010 8:17 AM
>> To: [hidden email]
>> Subject: Re: Zeiss or Olympus
>
>
>
> Mark
>
>> I was thinking about the use of GaAs ( gallium asrenide) detectors for
>> multiphoton by Zeiss for the NLO.  This are not a PMT and have very
>> different properties.
>
>> Dick Burry
>
>> Note: This message may contain confidential information. If this Email/Fax
>> has been sent to you by mistake, please notify the sender and delete it
>> immediately. Thank you.
>
>
>
>
>> --  >
>
>               **********************************************
>> 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.
>
> ________________________________
>
>> Spam
>> Not spam
>> Forget previous vote
>
>
> Richard W. Burry, Ph.D.
> Department of Neuroscience, College of Medicine
> Campus Microscopy and Imaging Facility, Director
> The Ohio State University
> Associate Editor, Journal of Histochemistry and Cytochemistry
> 277 Biomedical Research Tower
> 460 West Twelfth Avenue
> Columbus, Ohio 43210
> Voice 614.292.2814  Cell 614.638.3345  Fax [phone]
>
> --
>
>               **********************************************
> 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.

[George McNamara]

Find the money for an LSM 780 (NIH S10 maximum limit this year is
$600K and you can get a nicely equipped 780 within that amount - not
counting the MP laser, you can always move one of your lasers over).
If not, get an LSM710. The FV1000 is old technology.

Full disclosure: I manage an LSM 710 and just hosted a Zeiss
educational workshop featuring a 780 (www.zeiss.com/zoyc).

At 08:18 AM 3/9/2010, Vladimir Gukassyan wrote:

>Dear List Members,
>
>We're making a selection between Zeiss 710 and Olympus FV1000 for the
>multiphoton imaging.
>I would be thankful for all user experience cases and suggestions on
>this matter.
>
>Thank you in advance.
>With regards,
>Vladimir
>
>---------------------------------
>Vladimir Ghukasyan
>Confocal and Multiphoton Imaging Core,
>Neuroscience Research Center
>University of North Carolina
>115 Mason Farm Rd., Bld. 245, 7 Fl.
>Chapel Hill, NC 27599-7250
>Tel.: [phone]

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

[George McNamara]

Dear listserv,


GaAsP vs conventional PMT quantum efficiency curve is on the bottom of page 19 of the Zeiss LSM 780 Product Brochure (7.3 MB)  60-1-0015_lsm_780_e[1].pdf at
http://www.zeiss.de/C12567BE0045ACF1/Contents-Frame/0AE7CE3138743B6CC12576400050DF64

The 2010 Zeiss On Your Campus live cell imaging workshop CD (DVD?) "Single Molecules: Going Beyond Basic Imaging" pdf, page 16 (of 58) has silicon photodiode (APD), Ga-As-P PMT and Ga-As PMT QE curves (page 15 has CCD curves). If you are not attending/hosting a ZOYC, ask your local Zeiss rep for a copy of the CD or talk pdf's. The graphs in the ZOYC pdf look like molecular expressions figures, so you might also find them at M.E. or the M.E. hosted Zeiss web site.

As for Jim's mention that the GaAsP and conventional PMT cross-over at around 700 nm (maybe 670 nm in ZOYC graph): in almost 3 years at UM I cannot recall any user bringing a specimen with a fluorophore whose emission maximum longer than 700 nm. That said, a new paper, Tonninges et al 2010 J Microscopy has several new PTIR and  NeuroVue lipophilic dyes with nice 1p and 2p excitation and NIR emission properties. Paper is on neurotracing, but may also be useful as a DiI replacement for blood vessel painting if the price is right.


Sincerely,

George




At 04:28 PM 3/11/2010, you wrote:

From the Zeiss Brochure:

The new GaAsP detector technology is not confined to visual
light excitation. The LSM BiG (binary GaAsP) now also offers
you multicolor multiphoton imaging with GaAsP performance.
Two LSM BiG modules can be added to NLO systems
as transmitted and incident light NDDs, providing 4 ultrasensitive
detection channels. The LSM 780 NLO and LSM 710 NLO
let you penetrate deeper and detect more light.

So additional to the internal GaAsP spectral detector you can add a GaAsP NDD (BIG detector) for multiphoton imaging. And you can have the GaAsP single channel sitting directly over the objective. I think you can also couple two GaAsP detectors on an external port of the scan head

best wishes

Andreas

Thanks Andreas,

Does anyone have any idea of the maker or model number of these BiG detectors?

As I mentioned earlier, the count-rate performance of APD single-photon counting systems are limited by the capacitance of the sensor diode. This is in turn proportional to the the sensitive area of the detector.

For this reason, the sensitive areas tend to be small: maybe 100µm diameter. As this is much smaller than the ray bundle coming out the back of most high-mag, high-NA objectives, it is not obvious how one could "squeeze" the light bundle there to match the sensitive area. So I assume that the word "big" in your post is relevant and it would be nice to know how big and if it works in the counting mode, and whether it is an avalanche diode or merely a GaAsP photodiode (no amplification, but no multiplicative noise either. Very suitable for transmitted detectors....).

Cheers,

Jim P.

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

-----Original Message-----
From: RICHARD BURRY <[hidden email]>
To: [hidden email]
Sent: Wed, 10 Mar 2010 18:54
Subject: Re: Zeiss or Olympus
Jim
 
Thanks for the details of the GaAs and GaAsP detectors.  But my question remains, does Zeiss use either of these detectors in its 780 multiphoton?  And does this represent an improvement in S/N over standard PMTs?  My experience is with the NLO a while ago is that the GaAs detector was more sensitive but for samples with a range of emission intensity, it was difficult to not saturate some part of the sample.
 
Dick

----- Original Message -----
From: James Pawley <[hidden email]>
Date: Wednesday, March 10, 2010 11:58 am
Subject: Re: Zeiss or Olympus
To: [hidden email]

> Just to clarify, the 780 has a GaAsP (Gallium Arsenite Phosphate) detector, not GaAs, the difference in quantum efficiency can be seen e.g. in the Webb multiphoton review (Nature Biotechnology 2003, 21, 1369). The drawback is that GaAsP QE drops dramatically for wavelength > 700 nm, but they put a normal PMTs as the two additional channels on the 780, to cover the extended range. By the way GaAsP detectors are PMTs as well, it is just a different material of the photocathode, afterwards the photoelectrons are multiplied in the same way. GaAsP detectors reach 40% quantum efficiency which is about twice as sensitive as a normal PMT. APDs have 60-70% and a back-thinned CCD about 90%., so still a lot of signal is thrown away, not to mention the losses on the way to the detector.


> Andreas

> Hi all,


> Indeed, the GaAs and GaAs phosphide QE curves are very impressive. However, it is important to remember what is actually measured to make these curves. PMT curves refer to the fraction of photons striking the photocathode that produce photoelectrons (It is usually measured by allowing a calibrated amount of light to strike the photocathode and using a nano-ammeter to sense the total photoelectron current between the photocathode and all the other electrodes in the PMT). However, depending on the electrode geometry, 10-30% of these photoelectrons don't actually hit the first dynode (D1), and therefore do not contribute to the PMT output.


> Of those photoelectrons that do hit D1, a reasonable fraction fail to excite any secondary electrons, and again, do not contribute to the PMT output. There are many reasons for this but one is just Poisson statistics. If the average gain is say 4, then about 8% of the collisions will result in zero electrons being emitted. However, this effect is again multiplied by geometrical factors where SE produced in different parts of D1 have better or worse chances of actually striking D2 and producing a SE. Signal loss in this way depends a lot on the actual voltage between the photocathode and D1: it will be less when the voltage is higher. Unfortunately, few confocals seem to have been set up in such  way that this is always true. On average signal loss by failure to propagate after collision with D1 will be an additional 20-40%.


> Finally, the same type of Poisson effects that cause some signal to be lost entirely, cause the amount by which the remainder is amplified to be highly variable (10-90%). This variation degrades the accuracy of the output signal by introducing what is called multiplicative noise. Because this extra noise can only be "overcome" by counting more photons, its presence effectively reduces the effective QE of the device. In the best case, this reduction is about 40% and in the worst case (an exponential gain distribution, approximated by some micro PMTs) 75% (i.e., the QE is reduced to 60% or 25% of what it would have been if all photoelectrons were equally amplified).


> As a result, while the peak effective QE of a PMT with a GaAs or GaAsP photocathode is indeed much better than that of the more common S-20 photocathode, in terms of its effectiveness in providing an output current that is proportional to the input photon signal, the QE is more in the range of 3 -10% (depending on dynode geometry and first-dynode voltage) than 40%. (The 60% numbers are for APDs rather than for a GaAs or GaAsP photocathode on a PMT.)


> The performance can be improved somewhat on the few confocals that allow single-photon counting as this procedure eliminates multiplicative noise. (see below about the limitations imposed by photon counting)


> This tedious recital is I hope  justified by noting that, at least when EG&G was the major APD supplier, APD performance was not specified in terms of QE but as Photon Detection Efficiency (PDE). Although APDs can be operated in a low gain, proportional mode, their PDE under these conditions is very low (because APD multiplicative noise is very high and at low (non-avalanche breakdown) gain, by far the most likely gain of the initial photoelectron is zero).


> Therefore, high PDE (or high QE) AOD units tend to operate at high bias (high, avalanche gain) and this requires circuitry to quench the avalanche breakdown and count the single-photon pulses. Modern units contain both the sensor itself and the pulse counting and avalanche quenching circuits needed for counting the single-photon pulses. In other words (assuming that Hamamatsu follows the EG&G precedent), their QE figures for single-photon counting units already include any losses for non-propagation or multiplicative noise. Therefore, a quoted PID of 60% really does mean that 60% of the photons (of the specified wavelength) that strike the center of the active surface will be accurately counted.


> This is about 4-10x better than the performance of a similar GaAs or GaAsP photocathode on a PMT.


> This good news is tempered by the fact that, because of the high capacitance of the AOD itself, it is hard to count much faster than, say 30MHz. As 30MHz comes out to an absolute maximum of 60 counts during a 2 µs pixel, this means that at least 50% of your counts will be lost due to pulse pileup when 30 counts arrive per pixel and 10% will be lost at only 6 counts/pixel. In other words one has to be very careful to adjust the excitation intensity so as not to "clip" the brightness of those parts of the image that contain a lot of fluorophor.


> Lots more on this in The Handbook,


> 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.

> Just to mention, should one be stuck with PMTs instead GaAs,   one could play with the applied bias voltage to modify   dark noise   (to
  balance the  gain   versus noise.  )
Nice thing with Olympus Kalman filtering is   that its use would allow   increase the bias voltage of PMT 
 
 
 
 
Thanks
 
Axel   Central Imaging  (IMCB)  6-19B,  cell  +65 9271.5622
 

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of RICHARD BURRY
> Sent: Wednesday, March 10, 2010 8:17 AM
> To: [hidden email]
> Subject: Re: Zeiss or Olympus
 
Mark
 
> I was thinking about the use of GaAs ( gallium asrenide) detectors for multiphoton by Zeiss for the NLO.  This are not a PMT and have very different properties.
 
> Dick Burry

> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

> --  >
              **********************************************
> 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.

---

> Spam
> Not spam
> Forget previous vote


Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

-- 

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

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