Preamplifier for fast point-scanning

[Peter Rupprecht]

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Dear list,

I'm looking for preamplifiers for use in a resonant scanning 2P microscope, using fluorophors like GCaMP or OGB in vivo. Acquisition will be time-locked to the 80 MHz laser pulses, so the bandwidth of the PMT preamplifier should be at least 80 MHz in order not to smooth adjacent pixels too much.

Right now, I'm using this model, which seems to be used by many others in the field (as far as I can see):

http://www.femto.de/en/products/current-amplifiers/variable-gain-up-to-200-mhz-dhpca.html

Is there another option which might be better than this standard solution for some applications? I'd be glad to hear some opinions on that.

Best regards,
Peter

[Michael Giacomelli]

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Hi Peter,

I had hoped someone else would suggest parts because I am very curious
as well.  Right now I am using B&H ACA amplifiers for time-resolved
fluorescence measurements:

http://www.becker-hickl.com/pdf/Dbacal.pdf

These have very fast response times and good noise performance, as
well as low cost, but are sensitive to input/output loading and prone
to self-oscillation.  More annoyingly, they are AC coupled, meaning
that if you want to use them for intensity imaging, you will have to
perform more complex processing to extract the absolute intensity from
the pulse amplitude.

I would be interested to know what else is recommended as well.
Reading up on transimpedance amplifiers (and pulling a few apart), it
seems good design is sort of an art.

Mike

Mike

On Tue, Jan 27, 2015 at 4:40 AM, Peter Rupprecht
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear list,
>
> I'm looking for preamplifiers for use in a resonant scanning 2P microscope, using fluorophors like GCaMP or OGB in vivo. Acquisition will be time-locked to the 80 MHz laser pulses, so the bandwidth of the PMT preamplifier should be at least 80 MHz in order not to smooth adjacent pixels too much.
>
> Right now, I'm using this model, which seems to be used by many others in the field (as far as I can see):
>
> http://www.femto.de/en/products/current-amplifiers/variable-gain-up-to-200-mhz-dhpca.html
>
> Is there another option which might be better than this standard solution for some applications? I'd be glad to hear some opinions on that.
>
> Best regards,
> Peter

[Zdenek Svindrych-2]

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Hi Peter,
I tell you 80 MHz is a *high* frequency. What are you aiming at? 1024 x 1024
pixels at 80 fps? That sounds cool. But how many photons do you think you'll
get in a pixel? I bet mostly zero. Maybe one, from time to time... So you
may end up with heavy smoothing anyway. If you get whopping 10^9 photons per
second in the bright parts of your image, that translates to a SNR of 3.5 :-
).

I don't want to put you off, just go ahead and try it (and keep us posted).
Just remember that every piece of cable needs proper impedance matching
(termination), also the 'speed of light' is somewhat lower in a cable (20 cm
/ ns), also add some 1 to 5 ns per each comparator or amplifier stage and
you may end up not knowing which pulse came from which pixel (and of course
another 3 ns delay due to the lifetime of your fluorescent protein).

Detectors with integrated amplifiers are also available, but it's either low
speed 'intensity based', with some 6 MHz bandwidth, or fast photon counting
(and these are currently not able to do 10^9 counts per second). I haven't
seen anything in between that would be suitable for your project. So
combining fast detector, fast mplifier and fast digitizer is challenging,
even if you use commercially available building blocks...

I know I didn't help much, but, good luck!

zdenek


--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php



---------- Původní zpráva ----------
Od: Peter Rupprecht <[hidden email]>
Komu: [hidden email]
Datum: 27. 1. 2015 4:41:25
Předmět: Preamplifier for fast point-scanning

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Dear list,

I'm looking for preamplifiers for use in a resonant scanning 2P microscope,
using fluorophors like GCaMP or OGB in vivo. Acquisition will be time-locked
to the 80 MHz laser pulses, so the bandwidth of the PMT preamplifier should
be at least 80 MHz in order not to smooth adjacent pixels too much.

Right now, I'm using this model, which seems to be used by many others in
the field (as far as I can see):

http://www.femto.de/en/products/current-amplifiers/variable-gain-up-to-200-
mhz-dhpca.html

Is there another option which might be better than this standard solution
for some applications? I'd be glad to hear some opinions on that.

Best regards,
Peter"

[Craig Brideau]

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I recall using a 9000 series Hamamatsu amplifier with the H7422 tube, which
is sufficient for video rate imaging. I don't remember the precise
specifications but it should be pretty quick.

Craig
On Feb 3, 2015 12:03 AM, "Zdenek Svindrych" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Peter,
> I tell you 80 MHz is a *high* frequency. What are you aiming at? 1024 x
> 1024
> pixels at 80 fps? That sounds cool. But how many photons do you think
> you'll
> get in a pixel? I bet mostly zero. Maybe one, from time to time... So you
> may end up with heavy smoothing anyway. If you get whopping 10^9 photons
> per
> second in the bright parts of your image, that translates to a SNR of 3.5
> :-
> ).
>
> I don't want to put you off, just go ahead and try it (and keep us posted).
> Just remember that every piece of cable needs proper impedance matching
> (termination), also the 'speed of light' is somewhat lower in a cable (20
> cm
> / ns), also add some 1 to 5 ns per each comparator or amplifier stage and
> you may end up not knowing which pulse came from which pixel (and of course
> another 3 ns delay due to the lifetime of your fluorescent protein).
>
> Detectors with integrated amplifiers are also available, but it's either
> low
> speed 'intensity based', with some 6 MHz bandwidth, or fast photon counting
> (and these are currently not able to do 10^9 counts per second). I haven't
> seen anything in between that would be suitable for your project. So
> combining fast detector, fast mplifier and fast digitizer is challenging,
> even if you use commercially available building blocks...
>
> I know I didn't help much, but, good luck!
>
> zdenek
>
>
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> University of Virginia, Charlottesville, USA
> http://www.kcci.virginia.edu/workshop/index.php
>
>
>
> ---------- Původní zpráva ----------
> Od: Peter Rupprecht <[hidden email]>
> Komu: [hidden email]
> Datum: 27. 1. 2015 4:41:25
> Předmět: Preamplifier for fast point-scanning
>
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear list,
>
> I'm looking for preamplifiers for use in a resonant scanning 2P microscope,
> using fluorophors like GCaMP or OGB in vivo. Acquisition will be
> time-locked
> to the 80 MHz laser pulses, so the bandwidth of the PMT preamplifier should
> be at least 80 MHz in order not to smooth adjacent pixels too much.
>
> Right now, I'm using this model, which seems to be used by many others in
> the field (as far as I can see):
>
> http://www.femto.de/en/products/current-amplifiers/variable-gain-up-to-200-
> mhz-dhpca.html
>
> Is there another option which might be better than this standard solution
> for some applications? I'd be glad to hear some opinions on that.
>
> Best regards,
> Peter"
>

[Michael Giacomelli]

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*****

Hi Zdenek,

The refractive index (or vp in the case of a cable) specifies the
group velocity, not the group velocity dispersion (GVD), the latter
being the one that you care about.  You can have a lot of cabling and
amplification in your system so long as you are careful to manage GVD.
For our swept source optical coherence systems, for instance, we
measure femtosecond time of flight differences though tens or even
hundreds of nanoseconds of group delay.  We just very, very carefully
match the group delay between timing and signal arms while minimizing
the GVD.  If you look at some of the really high end transimpedance
amplifiers, they actually spec the group delay vs. frequency for this
reason.

I do something similar for FLIM measurements.

Mike

On Tue, Feb 3, 2015 at 2:01 AM, Zdenek Svindrych <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Peter,
> I tell you 80 MHz is a *high* frequency. What are you aiming at? 1024 x 1024
> pixels at 80 fps? That sounds cool. But how many photons do you think you'll
> get in a pixel? I bet mostly zero. Maybe one, from time to time... So you
> may end up with heavy smoothing anyway. If you get whopping 10^9 photons per
> second in the bright parts of your image, that translates to a SNR of 3.5 :-
> ).
>
> I don't want to put you off, just go ahead and try it (and keep us posted).
> Just remember that every piece of cable needs proper impedance matching
> (termination), also the 'speed of light' is somewhat lower in a cable (20 cm
> / ns), also add some 1 to 5 ns per each comparator or amplifier stage and
> you may end up not knowing which pulse came from which pixel (and of course
> another 3 ns delay due to the lifetime of your fluorescent protein).
>
> Detectors with integrated amplifiers are also available, but it's either low
> speed 'intensity based', with some 6 MHz bandwidth, or fast photon counting
> (and these are currently not able to do 10^9 counts per second). I haven't
> seen anything in between that would be suitable for your project. So
> combining fast detector, fast mplifier and fast digitizer is challenging,
> even if you use commercially available building blocks...
>
> I know I didn't help much, but, good luck!
>
> zdenek
>
>
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> University of Virginia, Charlottesville, USA
> http://www.kcci.virginia.edu/workshop/index.php
>
>
>
> ---------- Původní zpráva ----------
> Od: Peter Rupprecht <[hidden email]>
> Komu: [hidden email]
> Datum: 27. 1. 2015 4:41:25
> Předmět: Preamplifier for fast point-scanning
>
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear list,
>
> I'm looking for preamplifiers for use in a resonant scanning 2P microscope,
> using fluorophors like GCaMP or OGB in vivo. Acquisition will be time-locked
> to the 80 MHz laser pulses, so the bandwidth of the PMT preamplifier should
> be at least 80 MHz in order not to smooth adjacent pixels too much.
>
> Right now, I'm using this model, which seems to be used by many others in
> the field (as far as I can see):
>
> http://www.femto.de/en/products/current-amplifiers/variable-gain-up-to-200-
> mhz-dhpca.html
>
> Is there another option which might be better than this standard solution
> for some applications? I'd be glad to hear some opinions on that.
>
> Best regards,
> Peter"

[Vitaly Boyko]

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Dear All,
We are looking for a commercially available nuclear dye for fixed cells (tissue sections) in the near-IR portion of the spectrum that would be easily separated from Alexa-647, e.g with the excitation maxima in the 690-750 nm range?Any advice would be greatly appreciated.
Many thanks in advance.
Vitaly 

[Peter Rupprecht]

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*****

Hi,

Thanks for the replies!


Actually, I'm quite surprised that this fast PMT preamplifier seems to be rather uncommon. When somebody uses a 8 kHz resonant scanning confocal microscope at the lowest zoom, he might want to have not 512x512 pixels, but 2048x2048 pixels or even 4096x4096 pixels. That's not always useful for time-lapse imaging (as Zdenek pointed out), but very useful for taking a single picture or a z-stack with a lot of averaging. And to resolve 4096 pixels at a 8 kHz-scanned line (63 us for one line), you have to sample at ca 80 MHz.

Right now, I'm sampling at 80 MHz, and only when I do not need the full 4096 pixels, I bin it down in the software. But even for functional imaging, sometimes a very big picture - like 2048x512 - can be very useful.

Today, by coincidence, I called Becker&Hickl, and most likely I will test one of their ACA amplifiers side to side to the preamp that I'm using right now. (I was planning to take the 37dB preamp.) Becker&Hickl are specialized on photon counting technology, so this will be rather an abuse of their preamp. I will report the results in some weeks, if somebody is interested.

@Zdenek: A SNR of 3.5 or a little bit more might be enough for some applications ... I was planning to measure the photon count per pixel (using this method :
http://labrigger.com/blog/2010/07/30/measuring-the-gain-of-your-imaging-system/ ), but I always was busy with other things, so I cannot give you numbers for my imaging system.

@Craig: Are you possibly talking about this amplifier:
http://www.hamamatsu.com/jp/en/C9663/index.html ? Are there any drawbacks, and can you compare it to other amplifiers?

Michael wrote >> You can have a lot of cabling and amplification in your system so long as you are careful to manage GVD.

This is a little bit off-topic, but until now, I was simply sticking BNC cables with a 50 kOhm sign together, hoping that this would do it... however, I have the feeling that I get some lowpass-filtering / GVD of my detection signal. Do you have any tips for how to check and minimize the system's GVD?


Best,
Peter

[Michael Giacomelli]

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Hi Peter

The 37dB ACA model is worth trying, although its not really ideal.
Just be careful with source capacitance and cabling or you will get
oscillation because of the extremely high gain bandwidth product.  Was
really hoping someone would point out something better suited though.

GVD in typical 50 ohm RG-58 cables usually isn't a huge problem
because the attenuation is low enough below 1GHz and most timing
applications aren't sensitive at that level, so if you match cable
lengths, your pulse and timing will be relatively unaffected.  GVD
becomes problematic near frequencies with attenuation, so it tends to
be a bigger problem if you add filters to a system (or amplifiers with
too little bandwidth).  If you buy the ACA and intend to lowpass
filter it, look carefully at the minicircuits datasheets before
ordering, they are quite good about specing those things.  If you're
running impedance mismatched (e.g. 50kOhm terminating resistor), you
are just trading off bandwidth for a larger signal.

Mike


On Tue, Feb 3, 2015 at 2:22 PM, Peter Rupprecht
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi,
>
> Thanks for the replies!
>
>
> Actually, I'm quite surprised that this fast PMT preamplifier seems to be rather uncommon. When somebody uses a 8 kHz resonant scanning confocal microscope at the lowest zoom, he might want to have not 512x512 pixels, but 2048x2048 pixels or even 4096x4096 pixels. That's not always useful for time-lapse imaging (as Zdenek pointed out), but very useful for taking a single picture or a z-stack with a lot of averaging. And to resolve 4096 pixels at a 8 kHz-scanned line (63 us for one line), you have to sample at ca 80 MHz.
>
> Right now, I'm sampling at 80 MHz, and only when I do not need the full 4096 pixels, I bin it down in the software. But even for functional imaging, sometimes a very big picture - like 2048x512 - can be very useful.
>
> Today, by coincidence, I called Becker&Hickl, and most likely I will test one of their ACA amplifiers side to side to the preamp that I'm using right now. (I was planning to take the 37dB preamp.) Becker&Hickl are specialized on photon counting technology, so this will be rather an abuse of their preamp. I will report the results in some weeks, if somebody is interested.
>
> @Zdenek: A SNR of 3.5 or a little bit more might be enough for some applications ... I was planning to measure the photon count per pixel (using this method :
> http://labrigger.com/blog/2010/07/30/measuring-the-gain-of-your-imaging-system/ ), but I always was busy with other things, so I cannot give you numbers for my imaging system.
>
> @Craig: Are you possibly talking about this amplifier:
> http://www.hamamatsu.com/jp/en/C9663/index.html ? Are there any drawbacks, and can you compare it to other amplifiers?
>
> Michael wrote >> You can have a lot of cabling and amplification in your system so long as you are careful to manage GVD.
>
> This is a little bit off-topic, but until now, I was simply sticking BNC cables with a 50 kOhm sign together, hoping that this would do it... however, I have the feeling that I get some lowpass-filtering / GVD of my detection signal. Do you have any tips for how to check and minimize the system's GVD?
>
>
> Best,
> Peter

[Craig Brideau]

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On Tue, Feb 3, 2015 at 12:22 PM, Peter Rupprecht <[hidden email]>
wrote:

> *****
> @Craig: Are you possibly talking about this amplifier:
> http://www.hamamatsu.com/jp/en/C9663/index.html ? Are there any
> drawbacks, and can you compare it to other amplifiers?
>


Yes that's the one. It was neither worse nor better than the other
amplifiers I have encountered in commercial products, and it is
(relatively) inexpensive. You will need your own power supply for it, so
invest in a good quality, quiet supply. I used a pair of them with a very
early version of the Thorlabs resonant confocal scan head. I converted it
to a multiphoton and was able to get some decent calcium transient imaging
out of it.

Craig

[James Pawley]

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>
>@Zdenek: A SNR of 3.5 or a little bit more might be enough for some
>applications ... I was planning to measure the photon count per
>pixel (using this method :
>http://labrigger.com/blog/2010/07/30/measuring-the-gain-of-your-imaging-system/ 
>), but I always was busy with other things, so I cannot give you
>numbers for my imaging system.

Hi all,

Thanks to Labrigger for working on this important topic.

However, I have read his analysis and think that the assumption that
one can use this procedure to measure the number of photoelectrons
(PE: i.e., detected photons) created at the photocathode (PC) of the
PMT may be an over-simplification.

The analysis depends on the assumption that the only source of noise
in the data recorded in the "image" of a flat white field is Poisson
Noise associated with the small number of PEs produced at the
photocathode.  This might be true if PMTs were free from
multiplicative noise but in fact Poisson Noise also affects every
stage in the multiplication of a single PE after it leaves the PC. In
the very unusual case that the voltage between the PC and the first
dynode is 500-600 volts (and that this dynode has both the optimal
shape and the best GaAs surface), the gain of this stage may be 25
+/-5 or 20% additional noise. More commonly, this gain will be closer
to  4 +/-2 or 50% additional noise. More noise is added at each stage
and even though these noise terms are not additive (they are combined
as the sqrt of the sum of the squares), it is not at all uncommon for
this process to double or even triple the variation present in the
resulting signal beyond what one would expect from Poisson Noise
applied only to the number of PE. Furthermore, this added noise will
be somewhat larger if the system is working at a relatively high
signal level because then the PMT will be turned down, the gain/stage
correspondingly lower and the Poisson Noise proportionally higher.

Offsetting this error to some extent is the finite bandwidth of the
entire amplifier system (PMT plus the electronics between the final
dynode and the ADC). This bandwidth is in general unknown but may be
adjusted by the computer to more-or-less match what the computer
estimates is needed to pass the finest optical details that the
system can transmit on the basis of settings for wavelength,
objective NA, zoom/pixel size, and even PMT setting (high PMT voltage
implies a noisy signal that may benefit from the artificial,
1-dimensional smoothing that attends lower bandwidth).

Clearly, because bandwidth limits the maximum excursion that can be
transmitted between one pixel and its neighbour, it will tend to
reduce the apparent noise present in the digitized signal. The
magnitude of this clipping is unknown but may vary with the
parameters mentioned above.

This is relevant because, unlike the optical signal, the Poisson
Noise signal that we are searching for shows no correlation between
adjacent pixels. In particular, following the blog's suggestion of
using a high zoom (to reduce fixed pattern noise) may cause the
computer to limit the bandwidth more than using a lower zoom.

Although, as noted above, because these two factors bias the results
in opposite directions, their effects may cancel each other out to
some extent. However, we need to know a lot more about how the
components are actually operating before we can decide whether and to
what extent this is true.

The analysis also assumes that there is no fixed patterns noise in
the image of a "flat white field" as might be caused, for instance,
by field curvature, spherical aberration, vignetting, dust or other
optical parameters that may change detected signal across the field
of view.  I note that many of these sources of non-Poisson Noise can
be substantially reduced by recording two sequential frames and
obtaining a measure of the noise by subtracting one from the other.

For the analysis to work, it is also important to set the brightness
control (DC - offset) so that zero signal corresponds to closely to
zero intensity in the image memory.

I should note that multiplicative noise ceases to be a factor in
systems employing either hybrid PMT (where the first stage gain is
about 10,000) or effective photon-counting (i.e. a photon counting
where the recorded peak pixel signal is at least 10x smaller than the
saturation count rate of the system as set by pulse-pileup.).

One can avoid multiplcative noise by recording the data using  a CCD
(but NOT on an EM-CCD used with the electronic gain turned on) and
the record-two-then-subtract approach can again be used to reduce
inevitable fixed pattern noise.  However, this sensor will probably
work best when recording a fairly large signal (at least 10% of
peak?) so that read noise will be relatively insignificant. And as
above, the results will again be limited by the finite bandwidth of
the FET amplifier between the read-node and the ADC. Finally, when
using a CCD for quantitative measurements, it is particularly
important to remember that they are usually set up so that zero light
corresponds to 20-50 computer intensity units.

The noise performance of sCMOS detectors is both non-Gaussian and
depends strongly on the extent to which the internal pixel-by-pixel
variations in gain and offset are detected and corrected. This will
make their use for this type of measurement somewhat more difficult
unless the signal levels are well away from the noise floor.

Bottom line: Although the procedure may indeed give a useful
benchmark that we might call the "effective gain" of the signal path,
the measurement is subject to influence by a number of imaging
parameters and will not really allow one to measure how many
recorded-signal-intensity-units correspond to one PE.

Jim Pawley
--
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[Andrew York]

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 This is off-topic, but I suspect the same people who replied to this
thread could answer my question. Can someone describe the type of noise I
should expect with an Airyscan-style detector?

 I'm familiar with sCMOS and EMCCD cameras, but I have almost no experience
with megahertz few-pixel detectors. I assumed that the noise of each
'pixel' of the Airyscan detector would behave similarly to the pixels of a
sCMOS: Wavelength-dependent quantum efficiency above 50%, Poisson noise
that depends only on the number of photoelectrons generated by the signal
light, and additive Gaussian noise that depends only on the detector
settings. Is this true? Are there other important details I should know
about, like dynamic range, etc?

Thanks for the help!

-Andrew

On Wed, Feb 4, 2015 at 11:53 AM, James Pawley <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>
>> @Zdenek: A SNR of 3.5 or a little bit more might be enough for some
>> applications ... I was planning to measure the photon count per pixel
>> (using this method :
>> http://labrigger.com/blog/2010/07/30/measuring-the-gain-
>> of-your-imaging-system/ ), but I always was busy with other things, so I
>> cannot give you numbers for my imaging system.
>>
>
> Hi all,
>
> Thanks to Labrigger for working on this important topic.
>
> However, I have read his analysis and think that the assumption that one
> can use this procedure to measure the number of photoelectrons (PE: i.e.,
> detected photons) created at the photocathode (PC) of the PMT may be an
> over-simplification.
>
> The analysis depends on the assumption that the only source of noise in
> the data recorded in the "image" of a flat white field is Poisson Noise
> associated with the small number of PEs produced at the photocathode.  This
> might be true if PMTs were free from multiplicative noise but in fact
> Poisson Noise also affects every stage in the multiplication of a single PE
> after it leaves the PC. In the very unusual case that the voltage between
> the PC and the first dynode is 500-600 volts (and that this dynode has both
> the optimal shape and the best GaAs surface), the gain of this stage may be
> 25 +/-5 or 20% additional noise. More commonly, this gain will be closer
> to  4 +/-2 or 50% additional noise. More noise is added at each stage and
> even though these noise terms are not additive (they are combined as the
> sqrt of the sum of the squares), it is not at all uncommon for this process
> to double or even triple the variation present in the resulting signal
> beyond what one would expect from Poisson Noise applied only to the number
> of PE. Furthermore, this added noise will be somewhat larger if the system
> is working at a relatively high signal level because then the PMT will be
> turned down, the gain/stage correspondingly lower and the Poisson Noise
> proportionally higher.
>
> Offsetting this error to some extent is the finite bandwidth of the entire
> amplifier system (PMT plus the electronics between the final dynode and the
> ADC). This bandwidth is in general unknown but may be adjusted by the
> computer to more-or-less match what the computer estimates is needed to
> pass the finest optical details that the system can transmit on the basis
> of settings for wavelength, objective NA, zoom/pixel size, and even PMT
> setting (high PMT voltage implies a noisy signal that may benefit from the
> artificial, 1-dimensional smoothing that attends lower bandwidth).
>
> Clearly, because bandwidth limits the maximum excursion that can be
> transmitted between one pixel and its neighbour, it will tend to reduce the
> apparent noise present in the digitized signal. The magnitude of this
> clipping is unknown but may vary with the parameters mentioned above.
>
> This is relevant because, unlike the optical signal, the Poisson Noise
> signal that we are searching for shows no correlation between adjacent
> pixels. In particular, following the blog's suggestion of using a high zoom
> (to reduce fixed pattern noise) may cause the computer to limit the
> bandwidth more than using a lower zoom.
>
> Although, as noted above, because these two factors bias the results in
> opposite directions, their effects may cancel each other out to some
> extent. However, we need to know a lot more about how the components are
> actually operating before we can decide whether and to what extent this is
> true.
>
> The analysis also assumes that there is no fixed patterns noise in the
> image of a "flat white field" as might be caused, for instance, by field
> curvature, spherical aberration, vignetting, dust or other optical
> parameters that may change detected signal across the field of view.  I
> note that many of these sources of non-Poisson Noise can be substantially
> reduced by recording two sequential frames and obtaining a measure of the
> noise by subtracting one from the other.
>
> For the analysis to work, it is also important to set the brightness
> control (DC - offset) so that zero signal corresponds to closely to zero
> intensity in the image memory.
>
> I should note that multiplicative noise ceases to be a factor in systems
> employing either hybrid PMT (where the first stage gain is about 10,000) or
> effective photon-counting (i.e. a photon counting where the recorded peak
> pixel signal is at least 10x smaller than the saturation count rate of the
> system as set by pulse-pileup.).
>
> One can avoid multiplcative noise by recording the data using  a CCD (but
> NOT on an EM-CCD used with the electronic gain turned on) and the
> record-two-then-subtract approach can again be used to reduce inevitable
> fixed pattern noise.  However, this sensor will probably work best when
> recording a fairly large signal (at least 10% of peak?) so that read noise
> will be relatively insignificant. And as above, the results will again be
> limited by the finite bandwidth of the FET amplifier between the read-node
> and the ADC. Finally, when using a CCD for quantitative measurements, it is
> particularly important to remember that they are usually set up so that
> zero light corresponds to 20-50 computer intensity units.
>
> The noise performance of sCMOS detectors is both non-Gaussian and depends
> strongly on the extent to which the internal pixel-by-pixel variations in
> gain and offset are detected and corrected. This will make their use for
> this type of measurement somewhat more difficult unless the signal levels
> are well away from the noise floor.
>
> Bottom line: Although the procedure may indeed give a useful benchmark
> that we might call the "effective gain" of the signal path, the measurement
> is subject to influence by a number of imaging parameters and will not
> really allow one to measure how many recorded-signal-intensity-units
> correspond to one PE.
>
> Jim Pawley
> --
>            ****************************************
> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
> Canada, V0N3A0,
> Phone [phone], email <[hidden email]>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> 1-604-989-6146
>

[Michael Giacomelli]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Andrew,

The Airyscan system is a 32 channel GaAsP PMT array.  I don't believe
the datasheet for that part is publicly available, but there is
information on other GaAsP PMT array systems from Hamamatsu.  You
could look them up to get an idea what the dark counts, QE and
crosstalk are.  Looking at some of the ones on the Hamamatsu website,
their multichannel PMTs seem to have very similar QE and dark noise to
their single channel PMTs with similar cathode material.

Of course you may have additional noise because of the logic used to
read out the array and then reconstruct the wavefront.

Mike

On Tue, Feb 10, 2015 at 10:16 PM, Andrew York
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>  This is off-topic, but I suspect the same people who replied to this
> thread could answer my question. Can someone describe the type of noise I
> should expect with an Airyscan-style detector?
>
>  I'm familiar with sCMOS and EMCCD cameras, but I have almost no experience
> with megahertz few-pixel detectors. I assumed that the noise of each
> 'pixel' of the Airyscan detector would behave similarly to the pixels of a
> sCMOS: Wavelength-dependent quantum efficiency above 50%, Poisson noise
> that depends only on the number of photoelectrons generated by the signal
> light, and additive Gaussian noise that depends only on the detector
> settings. Is this true? Are there other important details I should know
> about, like dynamic range, etc?
>
> Thanks for the help!
>
> -Andrew
>
> On Wed, Feb 4, 2015 at 11:53 AM, James Pawley <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>>
>>> @Zdenek: A SNR of 3.5 or a little bit more might be enough for some
>>> applications ... I was planning to measure the photon count per pixel
>>> (using this method :
>>> http://labrigger.com/blog/2010/07/30/measuring-the-gain-
>>> of-your-imaging-system/ ), but I always was busy with other things, so I
>>> cannot give you numbers for my imaging system.
>>>
>>
>> Hi all,
>>
>> Thanks to Labrigger for working on this important topic.
>>
>> However, I have read his analysis and think that the assumption that one
>> can use this procedure to measure the number of photoelectrons (PE: i.e.,
>> detected photons) created at the photocathode (PC) of the PMT may be an
>> over-simplification.
>>
>> The analysis depends on the assumption that the only source of noise in
>> the data recorded in the "image" of a flat white field is Poisson Noise
>> associated with the small number of PEs produced at the photocathode.  This
>> might be true if PMTs were free from multiplicative noise but in fact
>> Poisson Noise also affects every stage in the multiplication of a single PE
>> after it leaves the PC. In the very unusual case that the voltage between
>> the PC and the first dynode is 500-600 volts (and that this dynode has both
>> the optimal shape and the best GaAs surface), the gain of this stage may be
>> 25 +/-5 or 20% additional noise. More commonly, this gain will be closer
>> to  4 +/-2 or 50% additional noise. More noise is added at each stage and
>> even though these noise terms are not additive (they are combined as the
>> sqrt of the sum of the squares), it is not at all uncommon for this process
>> to double or even triple the variation present in the resulting signal
>> beyond what one would expect from Poisson Noise applied only to the number
>> of PE. Furthermore, this added noise will be somewhat larger if the system
>> is working at a relatively high signal level because then the PMT will be
>> turned down, the gain/stage correspondingly lower and the Poisson Noise
>> proportionally higher.
>>
>> Offsetting this error to some extent is the finite bandwidth of the entire
>> amplifier system (PMT plus the electronics between the final dynode and the
>> ADC). This bandwidth is in general unknown but may be adjusted by the
>> computer to more-or-less match what the computer estimates is needed to
>> pass the finest optical details that the system can transmit on the basis
>> of settings for wavelength, objective NA, zoom/pixel size, and even PMT
>> setting (high PMT voltage implies a noisy signal that may benefit from the
>> artificial, 1-dimensional smoothing that attends lower bandwidth).
>>
>> Clearly, because bandwidth limits the maximum excursion that can be
>> transmitted between one pixel and its neighbour, it will tend to reduce the
>> apparent noise present in the digitized signal. The magnitude of this
>> clipping is unknown but may vary with the parameters mentioned above.
>>
>> This is relevant because, unlike the optical signal, the Poisson Noise
>> signal that we are searching for shows no correlation between adjacent
>> pixels. In particular, following the blog's suggestion of using a high zoom
>> (to reduce fixed pattern noise) may cause the computer to limit the
>> bandwidth more than using a lower zoom.
>>
>> Although, as noted above, because these two factors bias the results in
>> opposite directions, their effects may cancel each other out to some
>> extent. However, we need to know a lot more about how the components are
>> actually operating before we can decide whether and to what extent this is
>> true.
>>
>> The analysis also assumes that there is no fixed patterns noise in the
>> image of a "flat white field" as might be caused, for instance, by field
>> curvature, spherical aberration, vignetting, dust or other optical
>> parameters that may change detected signal across the field of view.  I
>> note that many of these sources of non-Poisson Noise can be substantially
>> reduced by recording two sequential frames and obtaining a measure of the
>> noise by subtracting one from the other.
>>
>> For the analysis to work, it is also important to set the brightness
>> control (DC - offset) so that zero signal corresponds to closely to zero
>> intensity in the image memory.
>>
>> I should note that multiplicative noise ceases to be a factor in systems
>> employing either hybrid PMT (where the first stage gain is about 10,000) or
>> effective photon-counting (i.e. a photon counting where the recorded peak
>> pixel signal is at least 10x smaller than the saturation count rate of the
>> system as set by pulse-pileup.).
>>
>> One can avoid multiplcative noise by recording the data using  a CCD (but
>> NOT on an EM-CCD used with the electronic gain turned on) and the
>> record-two-then-subtract approach can again be used to reduce inevitable
>> fixed pattern noise.  However, this sensor will probably work best when
>> recording a fairly large signal (at least 10% of peak?) so that read noise
>> will be relatively insignificant. And as above, the results will again be
>> limited by the finite bandwidth of the FET amplifier between the read-node
>> and the ADC. Finally, when using a CCD for quantitative measurements, it is
>> particularly important to remember that they are usually set up so that
>> zero light corresponds to 20-50 computer intensity units.
>>
>> The noise performance of sCMOS detectors is both non-Gaussian and depends
>> strongly on the extent to which the internal pixel-by-pixel variations in
>> gain and offset are detected and corrected. This will make their use for
>> this type of measurement somewhat more difficult unless the signal levels
>> are well away from the noise floor.
>>
>> Bottom line: Although the procedure may indeed give a useful benchmark
>> that we might call the "effective gain" of the signal path, the measurement
>> is subject to influence by a number of imaging parameters and will not
>> really allow one to measure how many recorded-signal-intensity-units
>> correspond to one PE.
>>
>> Jim Pawley
>> --
>>            ****************************************
>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
>> Canada, V0N3A0,
>> Phone [phone], email <[hidden email]>
>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
>> 1-604-989-6146
>>