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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:
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> 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:
>> *****
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>> 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 [log in to unmask]
>> 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)
>>>>> [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
|
