http://confocal-microscopy-list.275.s1.nabble.com/GaAsP-PMTs-tp5949611p5951627.html
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
unidirectional scanning mode). The Fukasawa 2006 IEEE paper (cited in
counts at zero). I used APD's on the X1 port of a Leica CW-STED in
Michalet 2008 (the latter 2 papers I found freely available on line). If
it works compared to our current FLIM PMTs and APDs. Plus, whoever buys
> *****
> 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 608-265-5315
>>>> 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/>>>
>>>
>