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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:
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> 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)
>> [log in to unmask]<mailto:[log in to unmask]>
>>
>> http://www.biology.missouri.edu/faculty/phillips.html
>> http://www.biotech.missouri.edu/mcc/
>>
>>
>> ----- Original Message -----
>> From: James Pawley<[log in to unmask]<mailto:[log in to unmask]>>
>> Date: Wednesday, March 10, 2010 11:58 am
>> Subject: Re: Zeiss or Olympus
>> To: [log in to unmask]<mailto:[log in to unmask]
>> >
>>
>>
>>> 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 [log in to unmask]<mailto:[log in to unmask]
>>> >
>>> 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)
>> [log in to unmask]<mailto:[log in to unmask]>
>>
>> http://www.biology.missouri.edu/faculty/phillips.html
>> http://www.biotech.missouri.edu/mcc/
>>
>>
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