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Re: Zeiss or Olympus

Posted by Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-or-Olympus-tp4702418p4714210.html

Re: Zeiss or Olympus

The other thing about APDs is that they are so vulnerable to excessive light that one bright pixel will trigger the safety circuit and the rest of your image is blank (QE = 0)!

Guy

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Thursday, 11 March 2010 3:58 AM
To: [hidden email]
Subject: 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

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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
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"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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"If it ain't diffraction, it must be statistics." Anon.

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