GaAsP PMTs

[Phillips, Thomas E.]

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While searching the confocal archive about GaAsP PMTs, I came across Jim Pawley's authoritative discussion (appended below but note that I took the liberty of highlighting one sentence in red) of why the real world QE of these PMTs might not really be 40% but I was left wondering just how much better are they than the conventional PMTs on a Zeiss or Leica confocal? Jim says they are "much better than that of the more common S-20 photocathode" . Is the ballpark sensitivity of a GaAsP unit about 2x higher? I would appreciate any insights or comments about the usefulness and limitations of these new detectors in core facilities. Tom

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]<mailto:[hidden email]>

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


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

> 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]<mailto:[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.





Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]<mailto:[hidden email]>

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/

[Mark Cannell]

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*****

Have a look here

http://jp.hamamatsu.com/resources/products/etd/eng/html/pmt_003.html

Cheers
On 22/01/2011, at 11:54 AM, Phillips, Thomas E. wrote:

> GaAsP

[George McNamara]

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Hi Tom,

See  see Wolfgang's MRT article at
http://onlinelibrary.wiley.com/doi/10.1002/jemt.20959/full

http://www.becker-hickl.de/pdf/hpm-appnote03.pdf        (pdf page 6 -
much larger area than an APD results in somewhat higher photon counts
... so much for simple QE curves! Example is from a confocal microscope
operate with 3 Airy Unit pinhole - difference may be even bigger with MP
excitation and non-descanned detection).
http://www.becker-hickl.de/pdf/dbhpm04.pdf
http://sales.hamamatsu.com/assets/pdf/catsandguides/p-dev_2007_TOTH0014E01.pdf     
(pdf page 8, bottom half)

If you have or are thinking of getting a Leica confocal, multiphoton,
and/or STED, ask your Leica rep for info on the HyD detectors -
available internally on the SP5, or NDD for MP, or on the X1 port (X1
usually uses APD's).

Enjoy,

George




On 1/21/2011 5:54 PM, Phillips, Thomas E. wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> While searching the confocal archive about GaAsP PMTs, I came across Jim Pawley's authoritative discussion (appended below but note that I took the liberty of highlighting one sentence in red) of why the real world QE of these PMTs might not really be 40% but I was left wondering just how much better are they than the conventional PMTs on a Zeiss or Leica confocal? Jim says they are "much better than that of the more common S-20 photocathode" . Is the ballpark sensitivity of a GaAsP unit about 2x higher? I would appreciate any insights or comments about the usefulness and limitations of these new detectors in core facilities. Tom
>
> Thomas E. Phillips, Ph.D
> Professor of Biological Sciences
> Director, Molecular Cytology Core
> 2 Tucker Hall
> University of Missouri
> Columbia, MO 65211-7400
> 573-882-4712 (office)
> 573-882-0123 (fax)
> [hidden email]<mailto:[hidden email]>
>
> http://www.biology.missouri.edu/faculty/phillips.html
> http://www.biotech.missouri.edu/mcc/
>
>
> ----- Original Message -----
> From: James Pawley<[hidden email]<mailto:[hidden email]>>
> Date: Wednesday, March 10, 2010 11:58 am
> Subject: Re: Zeiss or Olympus
> To: [hidden email]<mailto:[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
>>      
>    
>> 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]<mailto:[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.
>>      
>
>
>
>
> Thomas E. Phillips, Ph.D
> Professor of Biological Sciences
> Director, Molecular Cytology Core
> 2 Tucker Hall
> University of Missouri
> Columbia, MO 65211-7400
> 573-882-4712 (office)
> 573-882-0123 (fax)
> [hidden email]<mailto:[hidden email]>
>
> http://www.biology.missouri.edu/faculty/phillips.html
> http://www.biotech.missouri.edu/mcc/
>
>    

[Mark Cannell]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Before people get too carried away with these hybrid devices,  
detectors with APDs are non-linear at higher light levels. For  
confocal with (say) a 1 us dwell time this means you must arrange to  
have <10 photons per pixel. A second issue is gain loss with age in  
APDs although with most of the gain being provided by the cathode-APD  
acceleration voltage this may be less of an issue. This count rate  
limit may be overcome with array APDs but they introduce a loss of  
quantum efficiency and 'after pulsing' .  I guess what I am saying  
is  be careful in detector selection, they all have +/- points. But  
the improvement in QE for the new photocathodes is impressive (albeit  
at much higher dark count rates) .

Cheers Mark

On 23/01/2011, at 1:59 AM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Tom,
>
> See  see Wolfgang's MRT article at http://onlinelibrary.wiley.com/doi/10.1002/jemt.20959/full
>
> http://www.becker-hickl.de/pdf/hpm-appnote03.pdf        (pdf page 6  
> - much larger area than an APD results in somewhat higher photon  
> counts ... so much for simple QE curves! Example is from a confocal  
> microscope operate with 3 Airy Unit pinhole - difference may be even  
> bigger with MP excitation and non-descanned detection).
> http://www.becker-hickl.de/pdf/dbhpm04.pdf
> http://sales.hamamatsu.com/assets/pdf/catsandguides/p-dev_2007_TOTH0014E01.pdf 
>      (pdf page 8, bottom half)
>
> If you have or are thinking of getting a Leica confocal,  
> multiphoton, and/or STED, ask your Leica rep for info on the HyD  
> detectors - available internally on the SP5, or NDD for MP, or on  
> the X1 port (X1 usually uses APD's).
>
> Enjoy,
>
> George
>
>
>
>
> On 1/21/2011 5:54 PM, Phillips, Thomas E. wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> While searching the confocal archive about GaAsP PMTs, I came  
>> across Jim Pawley's authoritative discussion (appended below but  
>> note that I took the liberty of highlighting one sentence in red)  
>> of why the real world QE of these PMTs might not really be 40% but  
>> I was left wondering just how much better are they than the  
>> conventional PMTs on a Zeiss or Leica confocal? Jim says they are  
>> "much better than that of the more common S-20 photocathode" . Is  
>> the ballpark sensitivity of a GaAsP unit about 2x higher? I would  
>> appreciate any insights or comments about the usefulness and  
>> limitations of these new detectors in core facilities. Tom
>>
>> Thomas E. Phillips, Ph.D
>> Professor of Biological Sciences
>> Director, Molecular Cytology Core
>> 2 Tucker Hall
>> University of Missouri
>> Columbia, MO 65211-7400
>> 573-882-4712 (office)
>> 573-882-0123 (fax)
>> [hidden email]<mailto:[hidden email]>
>>
>> http://www.biology.missouri.edu/faculty/phillips.html
>> http://www.biotech.missouri.edu/mcc/
>>
>>
>> ----- Original Message -----
>> From: James Pawley<[hidden email]<mailto:[hidden email]>>
>> Date: Wednesday, March 10, 2010 11:58 am
>> Subject: Re: Zeiss or Olympus
>> To: [hidden email]<mailto:[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
>>>
>>
>>> 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]<mailto:[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.
>>>
>>
>>
>>
>>
>> Thomas E. Phillips, Ph.D
>> Professor of Biological Sciences
>> Director, Molecular Cytology Core
>> 2 Tucker Hall
>> University of Missouri
>> Columbia, MO 65211-7400
>> 573-882-4712 (office)
>> 573-882-0123 (fax)
>> [hidden email]<mailto:[hidden email]>
>>
>> http://www.biology.missouri.edu/faculty/phillips.html
>> http://www.biotech.missouri.edu/mcc/
>>
>>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Mark,

If 1 usec is too long, scan faster. The Leica resonant scanner operates
at 8000 lines per second. In 1,024 pixel/line mode, pixel dwell time is
approximately 0.125 usec (come to think of it, half that in
unidirectional scanning mode). The Fukasawa 2006 IEEE paper (cited in
Becker 2011) figure 7 shows that their (2006 era prototype) could
distinguish between 1, 2, 3, 4, 5 and 6 photons (and practically no
counts at zero). I used APD's on the X1 port of a Leica CW-STED in
September - I would love to see how well the hybrid detector(s) work
STED, especially with lifetime gating alluded to in Becker, Fukasawa and
Michalet 2008 (the latter 2 papers I found freely available on line). If
someone would please buy UM a B&H or Leica hybrid detector, I will work
to get it installed it on the NDD port (B&H) or in the SP5 scanhead
(HyD), hook it up to our SPC-830 TCSPC board, and let you know how well
it works compared to our current FLIM PMTs and APDs. Plus, whoever buys
it for us will have an excellent reason to come visit Miami to use it.

Enjoy,

George
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine


On 1/22/2011 3:29 PM, Mark Cannell wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Before people get too carried away with these hybrid devices,
> detectors with APDs are non-linear at higher light levels. For
> confocal with (say) a 1 us dwell time this means you must arrange to
> have <10 photons per pixel. A second issue is gain loss with age in
> APDs although with most of the gain being provided by the cathode-APD
> acceleration voltage this may be less of an issue. This count rate
> limit may be overcome with array APDs but they introduce a loss of
> quantum efficiency and 'after pulsing' .  I guess what I am saying  
> is  be careful in detector selection, they all have +/- points. But
> the improvement in QE for the new photocathodes is impressive (albeit
> at much higher dark count rates) .
>
> Cheers Mark
>
> On 23/01/2011, at 1:59 AM, George McNamara wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Tom,
>>
>> See  see Wolfgang's MRT article at
>> http://onlinelibrary.wiley.com/doi/10.1002/jemt.20959/full
>>
>> http://www.becker-hickl.de/pdf/hpm-appnote03.pdf        (pdf page 6 -
>> much larger area than an APD results in somewhat higher photon counts
>> ... so much for simple QE curves! Example is from a confocal
>> microscope operate with 3 Airy Unit pinhole - difference may be even
>> bigger with MP excitation and non-descanned detection).
>> http://www.becker-hickl.de/pdf/dbhpm04.pdf
>> http://sales.hamamatsu.com/assets/pdf/catsandguides/p-dev_2007_TOTH0014E01.pdf     
>> (pdf page 8, bottom half)
>>
>> If you have or are thinking of getting a Leica confocal, multiphoton,
>> and/or STED, ask your Leica rep for info on the HyD detectors -
>> available internally on the SP5, or NDD for MP, or on the X1 port (X1
>> usually uses APD's).
>>
>> Enjoy,
>>
>> George
>>
>>
>>
>>
>> On 1/21/2011 5:54 PM, Phillips, Thomas E. wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> While searching the confocal archive about GaAsP PMTs, I came across
>>> Jim Pawley's authoritative discussion (appended below but note that
>>> I took the liberty of highlighting one sentence in red) of why the
>>> real world QE of these PMTs might not really be 40% but I was left
>>> wondering just how much better are they than the conventional PMTs
>>> on a Zeiss or Leica confocal? Jim says they are "much better than
>>> that of the more common S-20 photocathode" . Is the ballpark
>>> sensitivity of a GaAsP unit about 2x higher? I would appreciate any
>>> insights or comments about the usefulness and limitations of these
>>> new detectors in core facilities. Tom
>>>
>>> Thomas E. Phillips, Ph.D
>>> Professor of Biological Sciences
>>> Director, Molecular Cytology Core
>>> 2 Tucker Hall
>>> University of Missouri
>>> Columbia, MO 65211-7400
>>> 573-882-4712 (office)
>>> 573-882-0123 (fax)
>>> [hidden email]<mailto:[hidden email]>
>>>
>>> http://www.biology.missouri.edu/faculty/phillips.html
>>> http://www.biotech.missouri.edu/mcc/
>>>
>>>
>>> ----- Original Message -----
>>> From: James Pawley<[hidden email]<mailto:[hidden email]>>
>>> Date: Wednesday, March 10, 2010 11:58 am
>>> Subject: Re: Zeiss or Olympus
>>> To:
>>> [hidden email]<mailto:[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
>>>>
>>>
>>>> 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]<mailto:[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.
>>>>
>>>
>>>
>>>
>>>
>>> Thomas E. Phillips, Ph.D
>>> Professor of Biological Sciences
>>> Director, Molecular Cytology Core
>>> 2 Tucker Hall
>>> University of Missouri
>>> Columbia, MO 65211-7400
>>> 573-882-4712 (office)
>>> 573-882-0123 (fax)
>>> [hidden email]<mailto:[hidden email]>
>>>
>>> http://www.biology.missouri.edu/faculty/phillips.html
>>> http://www.biotech.missouri.edu/mcc/
>>>
>>>
>

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[James Pawley]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Before people get too carried away with these
>hybrid devices, detectors with APDs are
>non-linear at higher light levels. For confocal
>with (say) a 1 us dwell time this means you must
>arrange to have <10 photons per pixel. A second
>issue is gain loss with age in APDs although
>with most of the gain being provided by the
>cathode-APD acceleration voltage this may be
>less of an issue. This count rate limit may be
>overcome with array APDs but they introduce a
>loss of quantum efficiency and 'after pulsing' .
>I guess what I am saying
>is  be careful in detector selection, they all
>have +/- points. But the improvement in QE for
>the new photocathodes is impressive (albeit at
>much higher dark count rates) .
>
>Cheers Mark

Hi all,

I echo Mark's cautions. There are long
discussions of these matters in Chapter 12 and
Appendix 3 of the Handbook. With respect to the
URL Mark sent out, ultra bialkali with a maximum
QE of about 43% looks very good BUT:

1) It occurs at a wavelength of 350 nm, well into
the near UV where we really seldom have need for
a detector in confocal-type micrsocopy.

2) Although no details are given, there is no
indication of how these curves were measured.
However, it is common to make such measurements
in terms of the current in nA leaving the
photocathode when a known flux of photons in a
given narrow wavelength band strikes it. The
ratio of the number of electrons/s in the current
to the photons/s in the light is the QE.

This sound good but:

        a) Not all photoelectrons leaving the PC,
actually strike the first dynode. The 20-30% that
do not, fail to multiply and this represents a
direct proportional loss of QE
        b) Not all of the PE that strike the
first dynode actually produce secondary
electrons. Partially this is just due to Poisson
noise: if the average first stage gain is only
say, 3, then for about 10% of arriving PEs, it
will be zero. It is actually more complex than
this and different parts of Dynode are likely to
have different SE coefficients. Again this lost
signal reduces the effective QE.
        c) Such QE curves usually represent the
best that can be obtained. However, as the PC
must be evaporated onto the inside of the glass
after each end-window tube has been evacuated and
pinched off, there is considerable variation in
the thickness and even the detailed atomic makeup
of this film (and hence it's QE: thicker PCs will
have higher QE in the red, lower in the blue).
Even selected tubes may have a QE 20% lower than
the published specs (i.e., maybe 37% rather than
43%) and  unselected tubes can be as much as 50%
less.

And then there is the matter of multiplicative
noise. Even on the best tubes set up in the best
way, (usually obtainable only when  voltage
between the PC and Dynode 1 is 5-10x higher than
that between the other sets of dynodes) this adds
20% to the Poisson noise, and can only be
"compensated for" by using 40% more signal in the
first place (Because Poisson Noise is
proportional to the sqrt of the signal, to
improve the S/N by a factor of 2, you must
increase the signal by a factor of 4). In other
words, the signal out the back of the PMT acts as
though the QE is only about 70% of what it would
have been after taking into account all of the
processes listed above.

Multiplicative noise can be substantially
eliminated by using pulse-counting circuitry, but
as Mark notes, pulse-counting tends to saturate
at the signal rates common in confocal microscopy
(i.e., the levels recorded in the brightest parts
of the image, (where the dye is) will be less
than they should be, perhaps much less.)

The reason for this tedious detail is that the
"QE" performance of avalanche photodiodes is not
usually measured in the same way (The exception
being so called linear-APDs). APDs have so much
multiplicative noise (and lost signal from PE
that don't avalanche) that they are almost always
used in a pulse-counting mode. As a result, the
"QE" performance of pulse-counting units is
usually measured in terms of Photon Detection
Efficiency  (PDE, there are other terms). A PDE
of 30% means that 30% of the photons of a given
wavelength that strike the detector will give
exactly one count in your image memory. Clearly a
PDE of 30% can give you a far more accurate
measure of the signal related to a given pixel
than one would get using a PMT having a raw QE of
30% but which is then subject to all the other
problems noted above.

But as Mark says, because APDs have to count
pulses, they are just not yet suitable for
"normal" confocal, where stain levels and other
variables mean that we are often surprised by
higher signals than can be handled by the
counting circuits.

Finally, the Subject line of this theme talks
about GaAsPMTs (but I could not find the first
post). GaAsPMTs are interesting because their
high-QE performance extents far into the red.
Unfortunately, this performance relies on being
able to create a PE using a low-energy photons
which in turn implies very dark current unless
the PC is either very small or is cooled (or both)

Cheers,

Jim Pawley

***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953              
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/            Applications due by March 15, 2011
               "If it ain't diffraction, it must be statistics." Anon.

>On 23/01/2011, at 1:59 AM, George McNamara wrote:
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>Hi Tom,
>>
>>See  see Wolfgang's MRT article at
>>http://onlinelibrary.wiley.com/doi/10.1002/jemt.20959/full
>>
>>http://www.becker-hickl.de/pdf/hpm-appnote03.pdf 
>>(pdf page 6 - much larger area than an APD
>>results in somewhat higher photon counts ... so
>>much for simple QE curves! Example is from a
>>confocal microscope operate with 3 Airy Unit
>>pinhole - difference may be even bigger with MP
>>excitation and non-descanned detection).
>>http://www.becker-hickl.de/pdf/dbhpm04.pdf
>>http://sales.hamamatsu.com/assets/pdf/catsandguides/p-dev_2007_TOTH0014E01.pdf 
>>(pdf page 8, bottom half)
>>
>>If you have or are thinking of getting a Leica
>>confocal, multiphoton, and/or STED, ask your
>>Leica rep for info on the HyD detectors -
>>available internally on the SP5, or NDD for MP,
>>or on the X1 port (X1 usually uses APD's).
>>
>>Enjoy,
>>
>>George
>>
>>
>>
>>
>>On 1/21/2011 5:54 PM, Phillips, Thomas E. wrote:
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>While searching the confocal archive about
>>>GaAsP PMTs, I came across Jim Pawley's
>>>authoritative discussion (appended below but
>>>note that I took the liberty of highlighting
>>>one sentence in red) of why the real world QE
>>>of these PMTs might not really be 40% but I
>>>was left wondering just how much better are
>>>they than the conventional PMTs on a Zeiss or
>>>Leica confocal? Jim says they are "much better
>>>than that of the more common S-20
>>>photocathode" . Is the ballpark sensitivity of
>>>a GaAsP unit about 2x higher? I would
>>>appreciate any insights or comments about the
>>>usefulness and limitations of these new
>>>detectors in core facilities. Tom
>>>
>>>Thomas E. Phillips, Ph.D
>>>Professor of Biological Sciences
>>>Director, Molecular Cytology Core
>>>2 Tucker Hall
>>>University of Missouri
>>>Columbia, MO 65211-7400
>>>573-882-4712 (office)
>>>573-882-0123 (fax)
>>>[hidden email]<mailto:[hidden email]>
>>>
>>>http://www.biology.missouri.edu/faculty/phillips.html
>>>http://www.biotech.missouri.edu/mcc/
>>>
>>>
>>>----- Original Message -----
>>>From: James Pawley<[hidden email]<mailto:[hidden email]>>
>>>Date: Wednesday, March 10, 2010 11:58 am
>>>Subject: Re: Zeiss or Olympus
>>>To:
>>>[hidden email]<mailto:[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
>>>>
>>>
>>>>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]<mailto:[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.
>>>>
>>>
>>>
>>>
>>>
>>>Thomas E. Phillips, Ph.D
>>>Professor of Biological Sciences
>>>Director, Molecular Cytology Core
>>>2 Tucker Hall
>>>University of Missouri
>>>Columbia, MO 65211-7400
>>>573-882-4712 (office)
>>>573-882-0123 (fax)
>>>[hidden email]<mailto:[hidden email]>
>>>
>>>http://www.biology.missouri.edu/faculty/phillips.html
>>>http://www.biotech.missouri.edu/mcc/

--
***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953              
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/            Applications due by March 15, 2011
               "If it ain't diffraction, it must be statistics." Anon.

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Jim,

If we had lots of light, there would be no reason to be looking at new
detectors.

With respect to 1, Fukasawa's Fig 3 shows GaAsP QD ~40% from 400 to
almost 700 nm (and "zero" above 750 nm - another plus with respect to
multiphoton excitation). Fukasawa and two coauthors are at Hamamatsu - I
am guessing they know how to measure QE.

With respect to 2a, no dynode on the hybrid detector. So, no
multiplicative noise. The device is also NOT an APD.

As for stain (fluorophore) levels - hopefully new detectors (hybrid or
other) will enable better use of direct labeled antibodies (or antibody
surrogates - see PubMed 20674470 if curious) and/or fluorescent protein
fusions expressed from endogenous promoters instead of massive
overexpression. This would enable better quantitation of the amount of
target molecules (simplest to achieve by countign single molecules). If
researchers start cutting back on fluorescent-phalloidin and DAPI,
Invitrogen will respond by changing to single use aliquots and shift
their profit making division from chemistry to packaging.

I encourage you to read Wolfgang's article. The Fukasawa and Michalet
articles were also nice reading.

Sincerely,

George


On 1/22/2011 5:24 PM, James Pawley wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Before people get too carried away with these hybrid devices,
>> detectors with APDs are non-linear at higher light levels. For
>> confocal with (say) a 1 us dwell time this means you must arrange to
>> have <10 photons per pixel. A second issue is gain loss with age in
>> APDs although with most of the gain being provided by the cathode-APD
>> acceleration voltage this may be less of an issue. This count rate
>> limit may be overcome with array APDs but they introduce a loss of
>> quantum efficiency and 'after pulsing' . I guess what I am saying  
>> is  be careful in detector selection, they all have +/- points. But
>> the improvement in QE for the new photocathodes is impressive (albeit
>> at much higher dark count rates) .
>>
>> Cheers Mark
>
> Hi all,
>
> I echo Mark's cautions. There are long discussions of these matters in
> Chapter 12 and Appendix 3 of the Handbook. With respect to the URL
> Mark sent out, ultra bialkali with a maximum QE of about 43% looks
> very good BUT:
>
> 1) It occurs at a wavelength of 350 nm, well into the near UV where we
> really seldom have need for a detector in confocal-type micrsocopy.
>
> 2) Although no details are given, there is no indication of how these
> curves were measured. However, it is common to make such measurements
> in terms of the current in nA leaving the photocathode when a known
> flux of photons in a given narrow wavelength band strikes it. The
> ratio of the number of electrons/s in the current to the photons/s in
> the light is the QE.
>
> This sound good but:
>
>     a) Not all photoelectrons leaving the PC, actually strike the
> first dynode. The 20-30% that do not, fail to multiply and this
> represents a direct proportional loss of QE
>     b) Not all of the PE that strike the first dynode actually produce
> secondary electrons. Partially this is just due to Poisson noise: if
> the average first stage gain is only say, 3, then for about 10% of
> arriving PEs, it will be zero. It is actually more complex than this
> and different parts of Dynode are likely to have different SE
> coefficients. Again this lost signal reduces the effective QE.
>     c) Such QE curves usually represent the best that can be obtained.
> However, as the PC must be evaporated onto the inside of the glass
> after each end-window tube has been evacuated and pinched off, there
> is considerable variation in the thickness and even the detailed
> atomic makeup of this film (and hence it's QE: thicker PCs will have
> higher QE in the red, lower in the blue). Even selected tubes may have
> a QE 20% lower than the published specs (i.e., maybe 37% rather than
> 43%) and  unselected tubes can be as much as 50% less.
>
> And then there is the matter of multiplicative noise. Even on the best
> tubes set up in the best way, (usually obtainable only when  voltage
> between the PC and Dynode 1 is 5-10x higher than that between the
> other sets of dynodes) this adds 20% to the Poisson noise, and can
> only be "compensated for" by using 40% more signal in the first place
> (Because Poisson Noise is proportional to the sqrt of the signal, to
> improve the S/N by a factor of 2, you must increase the signal by a
> factor of 4). In other words, the signal out the back of the PMT acts
> as though the QE is only about 70% of what it would have been after
> taking into account all of the processes listed above.
>
> Multiplicative noise can be substantially eliminated by using
> pulse-counting circuitry, but as Mark notes, pulse-counting tends to
> saturate at the signal rates common in confocal microscopy (i.e., the
> levels recorded in the brightest parts of the image, (where the dye
> is) will be less than they should be, perhaps much less.)
>
> The reason for this tedious detail is that the "QE" performance of
> avalanche photodiodes is not usually measured in the same way (The
> exception being so called linear-APDs). APDs have so much
> multiplicative noise (and lost signal from PE that don't avalanche)
> that they are almost always used in a pulse-counting mode. As a
> result, the "QE" performance of pulse-counting units is usually
> measured in terms of Photon Detection Efficiency  (PDE, there are
> other terms). A PDE of 30% means that 30% of the photons of a given
> wavelength that strike the detector will give exactly one count in
> your image memory. Clearly a PDE of 30% can give you a far more
> accurate measure of the signal related to a given pixel than one would
> get using a PMT having a raw QE of 30% but which is then subject to
> all the other problems noted above.
>
> But as Mark says, because APDs have to count pulses, they are just not
> yet suitable for "normal" confocal, where stain levels and other
> variables mean that we are often surprised by higher signals than can
> be handled by the counting circuits.
>
> Finally, the Subject line of this theme talks about GaAsPMTs (but I
> could not find the first post). GaAsPMTs are interesting because their
> high-QE performance extents far into the red. Unfortunately, this
> performance relies on being able to create a PE using a low-energy
> photons which in turn implies very dark current unless the PC is
> either very small or is cooled (or both)
>
> Cheers,
>
> Jim Pawley
>
> ***************************************************************************
>
> Prof. James B. Pawley,                Ph.  
> 608-238-3953                             21. N. Prospect Ave. Madison,
> WI 53726 USA [hidden email]
> 3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver
> Canada
> Info: http://www.3dcourse.ubc.ca/        Applications due by March 15,
> 2011
>            "If it ain't diffraction, it must be statistics." Anon.
>
>
>> On 23/01/2011, at 1:59 AM, George McNamara wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Tom,
>>>
>>> See  see Wolfgang's MRT article at
>>> http://onlinelibrary.wiley.com/doi/10.1002/jemt.20959/full
>>>
>>> http://www.becker-hickl.de/pdf/hpm-appnote03.pdf (pdf page 6 - much
>>> larger area than an APD results in somewhat higher photon counts ...
>>> so much for simple QE curves! Example is from a confocal microscope
>>> operate with 3 Airy Unit pinhole - difference may be even bigger
>>> with MP excitation and non-descanned detection).
>>> http://www.becker-hickl.de/pdf/dbhpm04.pdf
>>> http://sales.hamamatsu.com/assets/pdf/catsandguides/p-dev_2007_TOTH0014E01.pdf 
>>> (pdf page 8, bottom half)
>>>
>>> If you have or are thinking of getting a Leica confocal,
>>> multiphoton, and/or STED, ask your Leica rep for info on the HyD
>>> detectors - available internally on the SP5, or NDD for MP, or on
>>> the X1 port (X1 usually uses APD's).
>>>
>>> Enjoy,
>>>
>>> George
>>>
>>>
>>>
>>>
>>> On 1/21/2011 5:54 PM, Phillips, Thomas E. wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> While searching the confocal archive about GaAsP PMTs, I came
>>>> across Jim Pawley's authoritative discussion (appended below but
>>>> note that I took the liberty of highlighting one sentence in red)
>>>> of why the real world QE of these PMTs might not really be 40% but
>>>> I was left wondering just how much better are they than the
>>>> conventional PMTs on a Zeiss or Leica confocal? Jim says they are
>>>> "much better than that of the more common S-20 photocathode" . Is
>>>> the ballpark sensitivity of a GaAsP unit about 2x higher? I would
>>>> appreciate any insights or comments about the usefulness and
>>>> limitations of these new detectors in core facilities. Tom
>>>>
>>>> Thomas E. Phillips, Ph.D
>>>> Professor of Biological Sciences
>>>> Director, Molecular Cytology Core
>>>> 2 Tucker Hall
>>>> University of Missouri
>>>> Columbia, MO 65211-7400
>>>> 573-882-4712 (office)
>>>> 573-882-0123 (fax)
>>>> [hidden email]<mailto:[hidden email]>
>>>>
>>>> http://www.biology.missouri.edu/faculty/phillips.html
>>>> http://www.biotech.missouri.edu/mcc/
>>>>
>>>>
>>>> ----- Original Message -----
>>>> From: James Pawley<[hidden email]<mailto:[hidden email]>>
>>>> Date: Wednesday, March 10, 2010 11:58 am
>>>> Subject: Re: Zeiss or Olympus
>>>> To:
>>>> [hidden email]<mailto:[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
>>>>>
>>>>
>>>>> 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]<mailto:[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.
>>>>>
>>>>
>>>>
>>>>
>>>>
>>>> Thomas E. Phillips, Ph.D
>>>> Professor of Biological Sciences
>>>> Director, Molecular Cytology Core
>>>> 2 Tucker Hall
>>>> University of Missouri
>>>> Columbia, MO 65211-7400
>>>> 573-882-4712 (office)
>>>> 573-882-0123 (fax)
>>>> [hidden email]<mailto:[hidden email]>
>>>>
>>>> http://www.biology.missouri.edu/faculty/phillips.html
>>>> http://www.biotech.missouri.edu/mcc/
>
>

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Mark Cannell]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi George.

The hybrid device in question is most certainly a hybrid photocathode/  
APD. Look at Fig. 1. (without it a single stage vacuum photodiode does  
not have enough gain).

Cheers Mark

Regrds Mark
On 23/01/2011, at 12:32 PM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Jim,
>
> If we had lots of light, there would be no reason to be looking at  
> new detectors.
>
> With respect to 1, Fukasawa's Fig 3 shows GaAsP QD ~40% from 400 to  
> almost 700 nm (and "zero" above 750 nm - another plus with respect  
> to multiphoton excitation). Fukasawa and two coauthors are at  
> Hamamatsu - I am guessing they know how to measure QE.
>
> With respect to 2a, no dynode on the hybrid detector. So, no  
> multiplicative noise. The device is also NOT an APD.
>
> As for stain (fluorophore) levels - hopefully new detectors (hybrid  
> or other) will enable better use of direct labeled antibodies (or  
> antibody surrogates - see PubMed 20674470 if curious) and/or  
> fluorescent protein fusions expressed from endogenous promoters  
> instead of massive overexpression. This would enable better  
> quantitation of the amount of target molecules (simplest to achieve  
> by countign single molecules). If researchers start cutting back on  
> fluorescent-phalloidin and DAPI, Invitrogen will respond by changing  
> to single use aliquots and shift their profit making division from  
> chemistry to packaging.
>
> I encourage you to read Wolfgang's article. The Fukasawa and  
> Michalet articles were also nice reading.
>
> Sincerely,
>
> George
>
>
> On 1/22/2011 5:24 PM, James Pawley wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Before people get too carried away with these hybrid devices,  
>>> detectors with APDs are non-linear at higher light levels. For  
>>> confocal with (say) a 1 us dwell time this means you must arrange  
>>> to have <10 photons per pixel. A second issue is gain loss with  
>>> age in APDs although with most of the gain being provided by the  
>>> cathode-APD acceleration voltage this may be less of an issue.  
>>> This count rate limit may be overcome with array APDs but they  
>>> introduce a loss of quantum efficiency and 'after pulsing' . I  
>>> guess what I am saying  is  be careful in detector selection, they  
>>> all have +/- points. But the improvement in QE for the new  
>>> photocathodes is impressive (albeit at much higher dark count  
>>> rates) .
>>>
>>> Cheers Mark
>>
>> Hi all,
>>
>> I echo Mark's cautions. There are long discussions of these matters  
>> in Chapter 12 and Appendix 3 of the Handbook. With respect to the  
>> URL Mark sent out, ultra bialkali with a maximum QE of about 43%  
>> looks very good BUT:
>>
>> 1) It occurs at a wavelength of 350 nm, well into the near UV where  
>> we really seldom have need for a detector in confocal-type  
>> micrsocopy.
>>
>> 2) Although no details are given, there is no indication of how  
>> these curves were measured. However, it is common to make such  
>> measurements in terms of the current in nA leaving the photocathode  
>> when a known flux of photons in a given narrow wavelength band  
>> strikes it. The ratio of the number of electrons/s in the current  
>> to the photons/s in the light is the QE.
>>
>> This sound good but:
>>
>>    a) Not all photoelectrons leaving the PC, actually strike the  
>> first dynode. The 20-30% that do not, fail to multiply and this  
>> represents a direct proportional loss of QE
>>    b) Not all of the PE that strike the first dynode actually  
>> produce secondary electrons. Partially this is just due to Poisson  
>> noise: if the average first stage gain is only say, 3, then for  
>> about 10% of arriving PEs, it will be zero. It is actually more  
>> complex than this and different parts of Dynode are likely to have  
>> different SE coefficients. Again this lost signal reduces the  
>> effective QE.
>>    c) Such QE curves usually represent the best that can be  
>> obtained. However, as the PC must be evaporated onto the inside of  
>> the glass after each end-window tube has been evacuated and pinched  
>> off, there is considerable variation in the thickness and even the  
>> detailed atomic makeup of this film (and hence it's QE: thicker PCs  
>> will have higher QE in the red, lower in the blue). Even selected  
>> tubes may have a QE 20% lower than the published specs (i.e., maybe  
>> 37% rather than 43%) and  unselected tubes can be as much as 50%  
>> less.
>>
>> And then there is the matter of multiplicative noise. Even on the  
>> best tubes set up in the best way, (usually obtainable only when  
>> voltage between the PC and Dynode 1 is 5-10x higher than that  
>> between the other sets of dynodes) this adds 20% to the Poisson  
>> noise, and can only be "compensated for" by using 40% more signal  
>> in the first place (Because Poisson Noise is proportional to the  
>> sqrt of the signal, to improve the S/N by a factor of 2, you must  
>> increase the signal by a factor of 4). In other words, the signal  
>> out the back of the PMT acts as though the QE is only about 70% of  
>> what it would have been after taking into account all of the  
>> processes listed above.
>>
>> Multiplicative noise can be substantially eliminated by using pulse-
>> counting circuitry, but as Mark notes, pulse-counting tends to  
>> saturate at the signal rates common in confocal microscopy (i.e.,  
>> the levels recorded in the brightest parts of the image, (where the  
>> dye is) will be less than they should be, perhaps much less.)
>>
>> The reason for this tedious detail is that the "QE" performance of  
>> avalanche photodiodes is not usually measured in the same way (The  
>> exception being so called linear-APDs). APDs have so much  
>> multiplicative noise (and lost signal from PE that don't avalanche)  
>> that they are almost always used in a pulse-counting mode. As a  
>> result, the "QE" performance of pulse-counting units is usually  
>> measured in terms of Photon Detection Efficiency  (PDE, there are  
>> other terms). A PDE of 30% means that 30% of the photons of a given  
>> wavelength that strike the detector will give exactly one count in  
>> your image memory. Clearly a PDE of 30% can give you a far more  
>> accurate measure of the signal related to a given pixel than one  
>> would get using a PMT having a raw QE of 30% but which is then  
>> subject to all the other problems noted above.
>>
>> But as Mark says, because APDs have to count pulses, they are just  
>> not yet suitable for "normal" confocal, where stain levels and  
>> other variables mean that we are often surprised by higher signals  
>> than can be handled by the counting circuits.
>>
>> Finally, the Subject line of this theme talks about GaAsPMTs (but I  
>> could not find the first post). GaAsPMTs are interesting because  
>> their high-QE performance extents far into the red. Unfortunately,  
>> this performance relies on being able to create a PE using a low-
>> energy photons which in turn implies very dark current unless the  
>> PC is either very small or is cooled (or both)
>>
>> Cheers,
>>
>> Jim Pawley
>>
>> ***************************************************************************
>> Prof. James B. Pawley,                Ph.  
>> 608-238-3953                             21. N. Prospect Ave.  
>> Madison, WI 53726 USA [hidden email]
>> 3D Microscopy of Living Cells Course, June 11-23, 2011, UBC,  
>> Vancouver Canada
>> Info: http://www.3dcourse.ubc.ca/        Applications due by March  
>> 15, 2011
>>           "If it ain't diffraction, it must be statistics." Anon.
>>
>>
>>> On 23/01/2011, at 1:59 AM, George McNamara wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi Tom,
>>>>
>>>> See  see Wolfgang's MRT article at http://onlinelibrary.wiley.com/doi/10.1002/jemt.20959/full
>>>>
>>>> http://www.becker-hickl.de/pdf/hpm-appnote03.pdf (pdf page 6 -  
>>>> much larger area than an APD results in somewhat higher photon  
>>>> counts ... so much for simple QE curves! Example is from a  
>>>> confocal microscope operate with 3 Airy Unit pinhole - difference  
>>>> may be even bigger with MP excitation and non-descanned detection).
>>>> http://www.becker-hickl.de/pdf/dbhpm04.pdf
>>>> http://sales.hamamatsu.com/assets/pdf/catsandguides/p-dev_2007_TOTH0014E01.pdf 
>>>>  (pdf page 8, bottom half)
>>>>
>>>> If you have or are thinking of getting a Leica confocal,  
>>>> multiphoton, and/or STED, ask your Leica rep for info on the HyD  
>>>> detectors - available internally on the SP5, or NDD for MP, or on  
>>>> the X1 port (X1 usually uses APD's).
>>>>
>>>> Enjoy,
>>>>
>>>> George
>>>>
>>>>
>>>>
>>>>
>>>> On 1/21/2011 5:54 PM, Phillips, Thomas E. wrote:
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> While searching the confocal archive about GaAsP PMTs, I came  
>>>>> across Jim Pawley's authoritative discussion (appended below but  
>>>>> note that I took the liberty of highlighting one sentence in  
>>>>> red) of why the real world QE of these PMTs might not really be  
>>>>> 40% but I was left wondering just how much better are they than  
>>>>> the conventional PMTs on a Zeiss or Leica confocal? Jim says  
>>>>> they are "much better than that of the more common S-20  
>>>>> photocathode" . Is the ballpark sensitivity of a GaAsP unit  
>>>>> about 2x higher? I would appreciate any insights or comments  
>>>>> about the usefulness and limitations of these new detectors in  
>>>>> core facilities. Tom
>>>>>
>>>>> Thomas E. Phillips, Ph.D
>>>>> Professor of Biological Sciences
>>>>> Director, Molecular Cytology Core
>>>>> 2 Tucker Hall
>>>>> University of Missouri
>>>>> Columbia, MO 65211-7400
>>>>> 573-882-4712 (office)
>>>>> 573-882-0123 (fax)
>>>>> [hidden email]<mailto:[hidden email]>
>>>>>
>>>>> http://www.biology.missouri.edu/faculty/phillips.html
>>>>> http://www.biotech.missouri.edu/mcc/
>>>>>
>>>>>
>>>>> ----- Original Message -----
>>>>> From: James Pawley<[hidden email]<mailto:[hidden email]>>
>>>>> Date: Wednesday, March 10, 2010 11:58 am
>>>>> Subject: Re: Zeiss or Olympus
>>>>> To: [hidden email]<mailto:[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
>>>>>>
>>>>>
>>>>>> 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]<mailto:[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.
>>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> Thomas E. Phillips, Ph.D
>>>>> Professor of Biological Sciences
>>>>> Director, Molecular Cytology Core
>>>>> 2 Tucker Hall
>>>>> University of Missouri
>>>>> Columbia, MO 65211-7400
>>>>> 573-882-4712 (office)
>>>>> 573-882-0123 (fax)
>>>>> [hidden email]<mailto:[hidden email]>
>>>>>
>>>>> http://www.biology.missouri.edu/faculty/phillips.html
>>>>> http://www.biotech.missouri.edu/mcc/
>>
>>
>
>
> --
>
>
> George McNamara, PhD
> Analytical Imaging Core Facility
> University of Miami

[Stanislav Vitha]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

About 2 years ago, when I was building a 16-channel spectral detector for
our multiphoton microscope with the R5900U-xx-L16 linear PMT array as a
detector, I was told by Hammamatsu that for photon counting, I will be
better off using the original model with the standard bilalkali
photocathode, rather than using the newer super- or ultralakali models,
since the older model has much lower dark counts (and can be purchased as a
photon counting model R5900P-00-L16, tested for low-dark counts).
The peak QE around 400 nm was OK for our purpose, since besides fluorescent
proteins, we are interested in second harmonics signal from collagen, which
on our system is between 350 and 400 nm.

I wonder if there is going to be a hybrid multi-anode PMT.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257

On Sat, 22 Jan 2011 16:24:44 -0600, James Pawley <[hidden email]> wrote:

>
>I echo Mark's cautions. There are long
>discussions of these matters in Chapter 12 and
>Appendix 3 of the Handbook. With respect to the
>URL Mark sent out, ultra bialkali with a maximum
>QE of about 43% looks very good BUT:
>
>1) It occurs at a wavelength of 350 nm, well into
>the near UV where we really seldom have need for
>a detector in confocal-type micrsocopy.
>
>
>Cheers,
>
>Jim Pawley

[lgelman]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Thomas,

We host in our facility since 6 months a LSM710 with 2 "regular" confocal PMTs and 2 GaAsP detectors (the so-called BIG detectors). To make it short, we see with GFP or Alexa488 an improvement of a factor 2 with the GaAsPs in our signal: to get the same result, one would need to average (i.e. scan) twice with regular detectors when once is enough with the GaAsP. So, when the signal is week, the images are less noisy with the GaAsP. The more one "goes red", the bigger the difference. We got very nice images of an extremely faintly stained sample with Alexa647 with the GaAsPs, when we barely detected anything with the regular PMTs. I think you can grossly rely on the small graph given in the Zeiss brochure comparing the respective QE of both types of detectors, you will get there a good idea of how much you can improve your signal. Of course, you need to set detection windows with the regular PMTs in a way that you match those given by the filter cubes in front of the Big detectors to make any comparison. This is the limitation of these detectors in the LSM710: if your filters don't match your fluorophores, you can't do much (well, you can order new filters, which we did after a while to optimize detection of Alexa568 and Alexa633). Our users love the GaAsP detectors (and they love us too because we bought the GaAsP detectors :-))! I could not say anything about the dynamic range, as we did not test this (not really relevant in our case because we use the GaAsPs only when we have very faint signals). We'll see also how these detectors will age...

Hope that helps,

Best regards,

Laurent.


___________________________
Laurent Gelman, PhD
Friedrich Miescher Institut
Facility for Advanced Imaging and Microscopy
Head of Light Microscopy
WRO 1066.2.16
Maulbeerstrasse 66
CH-4058 Basel
[phone]





-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Phillips, Thomas E.
Sent: vendredi 21 janvier 2011 23:54
To: [hidden email]
Subject: GaAsP PMTs

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

While searching the confocal archive about GaAsP PMTs, I came across Jim Pawley's authoritative discussion (appended below but note that I took the liberty of highlighting one sentence in red) of why the real world QE of these PMTs might not really be 40% but I was left wondering just how much better are they than the conventional PMTs on a Zeiss or Leica confocal? Jim says they are "much better than that of the more common S-20 photocathode" . Is the ballpark sensitivity of a GaAsP unit about 2x higher? I would appreciate any insights or comments about the usefulness and limitations of these new detectors in core facilities. Tom

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]<mailto:[hidden email]>

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


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

> 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]<mailto:[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.





Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]<mailto:[hidden email]>

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/

[James Pawley]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>About 2 years ago, when I was building a 16-channel spectral detector for
>our multiphoton microscope with the R5900U-xx-L16 linear PMT array as a
>detector, I was told by Hammamatsu that for photon counting, I will be
>better off using the original model with the standard bilalkali
>photocathode, rather than using the newer super- or ultralakali models,
>since the older model has much lower dark counts (and can be purchased as a
>photon counting model R5900P-00-L16, tested for low-dark counts).
>The peak QE around 400 nm was OK for our purpose, since besides fluorescent
>proteins, we are interested in second harmonics signal from collagen, which
>on our system is between 350 and 400 nm.
>
>I wonder if there is going to be a hybrid multi-anode PMT.

Hi Stan,

A hybrid multi-anode PMT is an interesting idea. But I think that the
10-20kV voltages used on the hybrid PMT might not be easy to
implement given the small electrode geometry found on multi-anode
PMTs.

I am also interested in your comment on GaAsP PCs on linear array
PMT. I would have guessed that the PCs were small enough not to have
much signal but then I checked and the PC area of this model is 12.5
mm2 not 1mm2 as on some of the other linear models

Pulse counting is a relatively good idea for these multi-anode
detectors for a number of reasons.

1) the electron-multiplier part has very high multiplicative noise
(so you gain more effective QE by eliminating it).

2) as the total light signal is split between many parallel channels,
pulse-pileup is less of a problem.

3) their small size can result in short output pulses and therefore
fast count rates.

However, one must always be looking at the data rates to be sure that
pulse pileup is not a problem

Best

Jim Pawley

***************************************************************************
Prof. James B. Pawley,                          Ph.
608-238-3953                        
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/            Applications due by March 15, 2011
               "If it ain't diffraction, it must be statistics." Anon.

>Stan Vitha
>Microscopy and Imaging Center
>Texas A&M University
>BSBW 119
>College Station, TX 77843-2257
>
>On Sat, 22 Jan 2011 16:24:44 -0600, James Pawley <[hidden email]> wrote:
>
>>
>>I echo Mark's cautions. There are long
>>discussions of these matters in Chapter 12 and
>>Appendix 3 of the Handbook. With respect to the
>>URL Mark sent out, ultra bialkali with a maximum
>>QE of about 43% looks very good BUT:
>>
>>1) It occurs at a wavelength of 350 nm, well into
>>the near UV where we really seldom have need for
>>a detector in confocal-type micrsocopy.
>>
>>
>>Cheers,
>>
>>Jim Pawley

--