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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)
>>> [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/
>>>
>>>
>
--
George McNamara, PhD
Analytical Imaging Core Facility
University of Miami
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