Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-or-Olympus-tp4702418p4710640.html
Re: Zeiss or Olympus
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
Hi all,
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]
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.
Just to mention,
should one be stuck with PMTs instead GaAs, one could play
with the applied bias voltage to modify dark noise
(to
balance
the gain versus noise. )
Nice thing with
Olympus Kalman filtering is that its use would allow
increase the bias voltage of PMT
Thanks
Axel Central Imaging (IMCB) 6-19B, cell
+65 9271.5622
From: Confocal Microscopy List [[hidden email]] On Behalf Of RICHARD
BURRY
Sent: Wednesday, March 10, 2010 8:17 AM
To: [hidden email]
Subject: Re: Zeiss or Olympus
Mark
I was thinking about the use of GaAs ( gallium asrenide) detectors for
multiphoton by Zeiss for the NLO. This are not a PMT and have
very different properties.
Dick Burry
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**********************************************
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]
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.