Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-or-Olympus-tp4702418p4718473.html
Re: LSM 780 GaAsP
From the Zeiss Brochure:
The new GaAsP detector technology is not confined to visual
light excitation. The LSM BiG (binary GaAsP) now also offers
you multicolor multiphoton imaging with GaAsP performance.
Two LSM BiG modules can be added to NLO systems
as transmitted and incident light NDDs, providing 4 ultrasensitive
detection channels. The LSM 780 NLO and LSM 710 NLO
let you penetrate deeper and detect more light.
So additional to the internal GaAsP spectral detector
you can add a GaAsP NDD (BIG detector) for multiphoton imaging. And
you can have the GaAsP single channel sitting directly over the
objective. I think you can also couple two GaAsP detectors on an
external port of the scan head
best wishes
Andreas
Thanks Andreas,
Does anyone have any idea of the maker or model number of these
BiG detectors?
As I mentioned earlier, the count-rate performance of APD
single-photon counting systems are limited by the capacitance of the
sensor diode. This is in turn proportional to the the sensitive area
of the detector.
For this reason, the sensitive areas tend to be small: maybe
100µm diameter. As this is much smaller than the ray bundle coming out
the back of most high-mag, high-NA objectives, it is not obvious how
one could "squeeze" the light bundle there to match the
sensitive area. So I assume that the word "big" in your post
is relevant and it would be nice to know how big and if it works in
the counting mode, and whether it is an avalanche diode or merely a
GaAsP photodiode (no amplification, but no multiplicative noise
either. Very suitable for transmitted detectors....).
Cheers,
Jim P.
**********************************************
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.
-----Original Message-----
From: RICHARD BURRY <[hidden email]>
To: [hidden email]
Sent: Wed, 10 Mar 2010 18:54
Subject: Re: Zeiss or Olympus
Jim
Thanks for the details of the GaAs and GaAsP detectors. But
my question remains, does Zeiss use either of these detectors in its
780 multiphoton? And does this represent an improvement in
S/N over standard PMTs? My experience is with the NLO a
while ago is that the GaAs detector was more sensitive but for
samples with a range of emission intensity, it was difficult to not
saturate some part of the sample.
Dick
----- Original Message -----
From: James Pawley <[hidden email]>
Date: Wednesday, March 10, 2010 11:58 am
Subject: Re: Zeiss or Olympus
To: [hidden email]
> 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
[mailto:[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
> Note: This
message may contain confidential information. If this Email/Fax has
been sent to you by mistake, please notify the sender and delete it
immediately. Thank you.
>
-- >
**********************************************
> 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.
>
Spam
> Not
spam
> Forget previous
vote
Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814 Cell 614.638.3345 Fax
614.247.8849
--
**********************************************
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.