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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 [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
|
