confocal detectors and deconvolution

[James Pawley]

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Hi Zdenek,

Nice slide show reference! Thanks. I will steal some images!

However…

I think it is a shame that they do not really discuss the difference in “excess noise” between PMTs and SiPMs as this can affect effective QE very strongly.

Also, I think that they could have done a bit more to separate applications involving diffuse light from those involving almost-coherent light, like scanning microscopy, in which the signal originates from a very small volume (and therefore can be focussed onto any surface that is even slightly bigger). Of course, PMTs can have huge photocathodes (the R877-100 is 12,860 mm2) and, depending on their temp and IR sensitivity, can have very low dark count rates per square mm of photocathode. But in scanning light microscopy, there seems to be little need for many square mm of active area.

And even PMTs can have dark current problems. The first version of the Zeiss 510 used a large number (23? Don’t remember exactly.) of tiny servo-motors in which the windings were always excited. This heated the inside of the optics box enough to produce so many dark counts from their Hamamatsu PMT modules that they had to replace the servos with DC versions that were only excited when you changed some setting. And this was long before GaAsP photocathodes were common. From slide 19, you can see that the dark count rate of GaAsP is about 100x that of the more common bi-alkali PMTs.

All the same, silicon diodes do leak current at room temperature and so it is not surprising that most MPPCs (or SiPMs) are  supplied with Peltier cooling systems.

For most low-light, confocal applications it would seem that the S14420 series might work. It has a 1.5 x 1.5 mm array containing 900, 50µm cells with a fill factor of 81%, a PDE of 40% and a maximum dark count of 1,000 cps (or only 0.01 count per (long!) 10µs pixel).

https://www.hamamatsu.com/resources/pdf/ssd/s14420_series_kapd1061e.pdf

I know that a max PDE of 40% might sound less than the 40% QE on the plots of GaAsP PMT photocathodes, but these are different specs: PMT QE only refers to the fraction of photons making photoelectrons. About 30% of these photoelectrons fail to propagate down the electron-multiplier chain (i.e., they are lost to the signal). If you also include the fact that the MPPC has virtually no excess noise, this makes at least an additional 40% improvement in the effective QE.

BUT you have to make sure that the ray bundle fills the array.

(OK, maybe you don’t. Let’s assume that 600 of the 900 APDs on this device fall within a circle that just touches the sides of the square and that your signal ray-bundle just fills this circle (uniformly!) when your pinhole is open all the way. If we guess (from Slide 22 of the slide show) that the effective RC decay time is 40ns and choose a pixel time of 1 µs (or 25 decay times), a simple-minded analysis would lead us to conclude that a (massive) signal of 600 +/-24 detected photons/1 µs-pixel would lose close to 25 photon pulses due to pulse pile-up (i.e., a number similar the Poisson Noise uncertainty). A detected signal of 64 +/- 8 photons/1µs pixel from a darker area would lose less than one count, an error much smaller than the Poisson noise.

However, if you keep the same signal levels but use a pinhole that is 3.3 x smaller in diameter, the signal will now hit only 60 APDs and a 600 PE signal will lose 40% because of pulse pile-up. On the other hand, if the signal getting through the smaller pinhole is has gone down with the pinhole area and is now only 64 +/-8 pulses, then the loss is only 4%, or about 3 counts; smaller than the Poisson noise. For 10µs pixels, the signal would go up by 10 times but the percentage loss would be the same..

So using this detector, you would have another thing to worry about: “At this signal level and this pinhole size, am I losing signal to pileup?”

Annoying, but a geometric factor that the computer could easily keep track of and warn you when there was a problem.

A worse problem is that, if the bundle diameter is larger than the back-projected image of the Airy Disk, the light in the bundle is NOT evenly distributed  As long as there isn’t much spherical aberration, the image of the spot at the pinhole is always a LOT brighter in the centre. So this might be a potential problem, one that could be reduced by using a larger array (3x3 or 6x6 mm) and changing the optics to fill these arrays. Larger arrays cost more and would increase dark counts by about 4x or 16x (resp.) but probably still not enough to worry about.

Happy designing.

Jim Pawley

              ****************************************
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]<mailto:[hidden email]>>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

On May 14, 18, at 9:23 AM, [hidden email]<mailto:[hidden email]> wrote:

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To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call them),
note that their dark noise is some 3 - 4 orders of magnitude higher than
that of regular PMTs! Of course, chilling the detector to -80 degC (which is
common with EMCCDs, for example) would solve this issue, but the cost would
be prohibitive...
For more details on SiPMs vs PMTs see here:

https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB


Cheers, zdenek



---------- Původní e-mail ----------
Od: JAMES B PAWLEY <[hidden email]<mailto:[hidden email]>>
Komu: [hidden email]<mailto:[hidden email]>
Datum: 14. 5. 2018 0:27:35
Předmět: Re: confocal detectors and deconvolution
"*****
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Hi all,

I would like to Eecho Michael’s points.

Because hybrid photodetectors have a very high gain (>>1,000x) in their
first stage, they produce very little “excess noise” (also called
Multiplicative Noise). As a result, it is possible to characterize their
output as "25 photons detected” (although it might be safer to think of it
as "25 photons detected this time" or ”25 +/- 5 photons.").

Straight PMTs do not share this feature and because single photoelectrons
produce output pulses that vary significantly in size, even the very best
PMTs produce an uncertainty in the magnitude of the signal presented to the
ADC that is at least 40% larger in relative terms than would be the case in
the absence of this excess noise. On PMTs having electron multipliers
optimized for other reasons (such as making them very small, like those in
the 32-PMT linear arrays), the increase in uncertainty is closer to 100% (i.
e., The signal has the same uncertainty that it would have if 4 times fewer
photons were counted perfectly.)

Either type of PMT can have a GaAsP photocathode but it will need to be
cooled.

Although single APDs may have a high photon detection efficiency (PDE, a
spec that is like QE but which includes the signal lost by photoelectrons
that do not avalanche at all) they have such massive excess noise that it is
essential to use them with pulse-counting circuits and these circuits are
just too slow for use in beam-scanning light microscopy.

The solution is the multi-pixel photon counter (MPPC, a development from the
SPAD (single photon avalanche device),

https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf

https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_
ultimate_photosensor/index.html ).

The surface of an MPPC is covered with an array of 400 to 20,000 APDs, each
connected to the high-voltage rail through its own damping resister. The
resistor causes the voltage across the APD to drop as the avalanche
proceeds. This quenches the discharge and produces single-photon pulses of
very uniform size. As all the APDs are electrically in parallel, these
single-photon current pulses simply add up producing an output current
signal almost devoid of excess noise. What could be better? And in addition
they are significantly less sensitive than hybrid PMTs to overheating damage
if accidentally exposed to a bright light.

There are of course limitations: 1) A significant fraction (20-40%) of the
MPPC's surface is taken up with the resistors and the wiring to provide each
APD with + and - voltages. Photons absorbed or reflected in these areas are
lost. 2) The system is only free of pulse-pileup losses to the extent that
no APD absorbs more than one photon within its RC relaxation time (set by
the R of the resistor, and the capacitance (C) of the sensitive area of the
APD. Larger individual APDs “waste” proportionally fewer photons hitting the
resistor and wiring, (increasing their effective QE) but this increases
their capacitance (making them more susceptible to pulse-pileup).

All will be well as long as the number of photons absorbed in the active
areas during time period RC is small (5%?) compared to the number of APDs in
the array THAT ARE ILLUMINATED BY THE BEAM.

The caps above are to remind everyone that, to work properly, the size of
the ray bundle striking the MPPC must be matched to the size of the APD
array (1.3 to 6 mm square). This can be a problem if we imagine the ray
bundle being limited by a confocal aperture that can be varied in size over
a substantial range. (Do we need a zoom lens to make all possible signal ray
-bundles match the size of the MPPC array?)

Apart from this, I would like to second the comment that deep imaging is
usually limited by spherical aberration and systems that can correct for
this without “bumping the specimen while you try to adjust the collar” are
to be preferred.

I would also like to reaffirm that, assuming the pixel size meets Nyquist,
you should ONLY evaluate results after deconvolving the data with an
appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist-
sampled image will be at least 4 (more likely 5) pixels wide, all the noise
terms affect single pixel values. i.e., they have frequency components at
least 4x higher than that which can represent any real structure. In scanned
fluorescent imaging, one deconvolves more to reduce noise than to increase “
spatial resolution” (although you can also increase resolution as long as
you have massive amounts of signal.)

Cheers,

Jim Pawley
****************************************
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
Canada, V0N3A0,
Phone [phone], email <[hidden email]<mailto:[hidden email]><mailto:[hidden email]>>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146


On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:[hidden email]><mailto:mmodel@
KENT.EDU<http://KENT.EDU>>> wrote:

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

We have potential users who want to quantify some kind of small aggregates
in the brain. I am afraid that deconvolution can make noise look like such
aggregates. Perhaps collecting a noisy image twice and comparing two
deconvolved images might help, but that seems too much work. Am I wrong?

-----Original Message-----
From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> On Behalf
Of Steffen Dietzel
Sent: Friday, May 11, 2018 10:49 AM
To: [hidden email]<mailto:[hidden email]>
Subject: Re: confocal detectors and deconvolution

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Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
there is no big difference between HyDs and GaAsP detectors.

I disagree on this. In my view, the major difference is that the HyD always
operates in photon counting mode whether, as far as I know, the PMTs (with
or without GaAsP) create an electron cloud of which the size is determined
by the number of photoelectrons AND statistics, and the cloud size is then
digitized. So the output created by one photon may vary substantially
depending on the number of electrons created on the first dynodes (which in
turn is a statistical process). My information may be outdated and newer
PMTs might have extra tricks, if so please correct me.

Another difference is apparently the size of the photcathode. If memory
serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs)
creates more dark noise. I like our HyDs a lot, I appreciate having a gray
value of "21 photons" instead of some random number. But having said this,
at the end of the day what counts is the sensitivity of the whole system,
and not of the detector alone. So to do this right there is no substitute
for testing your own samples on different machines with your applications in
mind.

As for deconvolution, yes, it can create artefacts. But so does confocal
microscopy (a point becomes an Airy pattern, not a point). And if you do it
right the deconvolved image will be closer to the truth than the original
image. Should you have the third edition of the handbook around, have a look
at the preface, last paragraph.

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

"

[Michael Giacomelli]

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*****

Hi James,

That is a very interesting post.  I've read about SiPMs before, but
did not realize they were so far as long. The need to evenly
illuminate a large area is slightly annoying (probably not going to
work well for non-descanned 2P), but could be designed around for a
descanned system as you suggest.  The specs certainly look
interesting, especially the decreased sensitivity to strong light, and
the very low multiplicative noise.

I wonder if anyone has tried one in a scanning confocal or 2p system
and compared to conventional PMTs (e.g. h7422s or similar)?  I did a
quick search, but didn't see many papers using them for conventional
confocal scanning.

Mike

On Mon, May 14, 2018 at 10:50 PM, JAMES B 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.
> *****
>
> Hi Zdenek,
>
> Nice slide show reference! Thanks. I will steal some images!
>
> However…
>
> I think it is a shame that they do not really discuss the difference in “excess noise” between PMTs and SiPMs as this can affect effective QE very strongly.
>
> Also, I think that they could have done a bit more to separate applications involving diffuse light from those involving almost-coherent light, like scanning microscopy, in which the signal originates from a very small volume (and therefore can be focussed onto any surface that is even slightly bigger). Of course, PMTs can have huge photocathodes (the R877-100 is 12,860 mm2) and, depending on their temp and IR sensitivity, can have very low dark count rates per square mm of photocathode. But in scanning light microscopy, there seems to be little need for many square mm of active area.
>
> And even PMTs can have dark current problems. The first version of the Zeiss 510 used a large number (23? Don’t remember exactly.) of tiny servo-motors in which the windings were always excited. This heated the inside of the optics box enough to produce so many dark counts from their Hamamatsu PMT modules that they had to replace the servos with DC versions that were only excited when you changed some setting. And this was long before GaAsP photocathodes were common. From slide 19, you can see that the dark count rate of GaAsP is about 100x that of the more common bi-alkali PMTs.
>
> All the same, silicon diodes do leak current at room temperature and so it is not surprising that most MPPCs (or SiPMs) are  supplied with Peltier cooling systems.
>
> For most low-light, confocal applications it would seem that the S14420 series might work. It has a 1.5 x 1.5 mm array containing 900, 50µm cells with a fill factor of 81%, a PDE of 40% and a maximum dark count of 1,000 cps (or only 0.01 count per (long!) 10µs pixel).
>
> https://www.hamamatsu.com/resources/pdf/ssd/s14420_series_kapd1061e.pdf
>
> I know that a max PDE of 40% might sound less than the 40% QE on the plots of GaAsP PMT photocathodes, but these are different specs: PMT QE only refers to the fraction of photons making photoelectrons. About 30% of these photoelectrons fail to propagate down the electron-multiplier chain (i.e., they are lost to the signal). If you also include the fact that the MPPC has virtually no excess noise, this makes at least an additional 40% improvement in the effective QE.
>
> BUT you have to make sure that the ray bundle fills the array.
>
> (OK, maybe you don’t. Let’s assume that 600 of the 900 APDs on this device fall within a circle that just touches the sides of the square and that your signal ray-bundle just fills this circle (uniformly!) when your pinhole is open all the way. If we guess (from Slide 22 of the slide show) that the effective RC decay time is 40ns and choose a pixel time of 1 µs (or 25 decay times), a simple-minded analysis would lead us to conclude that a (massive) signal of 600 +/-24 detected photons/1 µs-pixel would lose close to 25 photon pulses due to pulse pile-up (i.e., a number similar the Poisson Noise uncertainty). A detected signal of 64 +/- 8 photons/1µs pixel from a darker area would lose less than one count, an error much smaller than the Poisson noise.
>
> However, if you keep the same signal levels but use a pinhole that is 3.3 x smaller in diameter, the signal will now hit only 60 APDs and a 600 PE signal will lose 40% because of pulse pile-up. On the other hand, if the signal getting through the smaller pinhole is has gone down with the pinhole area and is now only 64 +/-8 pulses, then the loss is only 4%, or about 3 counts; smaller than the Poisson noise. For 10µs pixels, the signal would go up by 10 times but the percentage loss would be the same..
>
> So using this detector, you would have another thing to worry about: “At this signal level and this pinhole size, am I losing signal to pileup?”
>
> Annoying, but a geometric factor that the computer could easily keep track of and warn you when there was a problem.
>
> A worse problem is that, if the bundle diameter is larger than the back-projected image of the Airy Disk, the light in the bundle is NOT evenly distributed  As long as there isn’t much spherical aberration, the image of the spot at the pinhole is always a LOT brighter in the centre. So this might be a potential problem, one that could be reduced by using a larger array (3x3 or 6x6 mm) and changing the optics to fill these arrays. Larger arrays cost more and would increase dark counts by about 4x or 16x (resp.) but probably still not enough to worry about.
>
> Happy designing.
>
> Jim Pawley
>
>               ****************************************
> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0,
> Phone [phone], email <[hidden email]<mailto:[hidden email]>>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>
> On May 14, 18, at 9:23 AM, [hidden email]<mailto:[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<http://www.imgur.com/> and include the link in your posting.
> *****
>
>
> To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call them),
> note that their dark noise is some 3 - 4 orders of magnitude higher than
> that of regular PMTs! Of course, chilling the detector to -80 degC (which is
> common with EMCCDs, for example) would solve this issue, but the cost would
> be prohibitive...
> For more details on SiPMs vs PMTs see here:
>
> https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB
>
>
> Cheers, zdenek
>
>
>
> ---------- Původní e-mail ----------
> Od: JAMES B PAWLEY <[hidden email]<mailto:[hidden email]>>
> Komu: [hidden email]<mailto:[hidden email]>
> Datum: 14. 5. 2018 0:27:35
> Předmět: Re: confocal detectors and deconvolution
> "*****
> 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<http://www.imgur.com/> and include the link in your posting.
> *****
>
> Hi all,
>
> I would like to Eecho Michael’s points.
>
> Because hybrid photodetectors have a very high gain (>>1,000x) in their
> first stage, they produce very little “excess noise” (also called
> Multiplicative Noise). As a result, it is possible to characterize their
> output as "25 photons detected” (although it might be safer to think of it
> as "25 photons detected this time" or ”25 +/- 5 photons.").
>
> Straight PMTs do not share this feature and because single photoelectrons
> produce output pulses that vary significantly in size, even the very best
> PMTs produce an uncertainty in the magnitude of the signal presented to the
> ADC that is at least 40% larger in relative terms than would be the case in
> the absence of this excess noise. On PMTs having electron multipliers
> optimized for other reasons (such as making them very small, like those in
> the 32-PMT linear arrays), the increase in uncertainty is closer to 100% (i.
> e., The signal has the same uncertainty that it would have if 4 times fewer
> photons were counted perfectly.)
>
> Either type of PMT can have a GaAsP photocathode but it will need to be
> cooled.
>
> Although single APDs may have a high photon detection efficiency (PDE, a
> spec that is like QE but which includes the signal lost by photoelectrons
> that do not avalanche at all) they have such massive excess noise that it is
> essential to use them with pulse-counting circuits and these circuits are
> just too slow for use in beam-scanning light microscopy.
>
> The solution is the multi-pixel photon counter (MPPC, a development from the
> SPAD (single photon avalanche device),
>
> https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf
>
> https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_
> ultimate_photosensor/index.html ).
>
> The surface of an MPPC is covered with an array of 400 to 20,000 APDs, each
> connected to the high-voltage rail through its own damping resister. The
> resistor causes the voltage across the APD to drop as the avalanche
> proceeds. This quenches the discharge and produces single-photon pulses of
> very uniform size. As all the APDs are electrically in parallel, these
> single-photon current pulses simply add up producing an output current
> signal almost devoid of excess noise. What could be better? And in addition
> they are significantly less sensitive than hybrid PMTs to overheating damage
> if accidentally exposed to a bright light.
>
> There are of course limitations: 1) A significant fraction (20-40%) of the
> MPPC's surface is taken up with the resistors and the wiring to provide each
> APD with + and - voltages. Photons absorbed or reflected in these areas are
> lost. 2) The system is only free of pulse-pileup losses to the extent that
> no APD absorbs more than one photon within its RC relaxation time (set by
> the R of the resistor, and the capacitance (C) of the sensitive area of the
> APD. Larger individual APDs “waste” proportionally fewer photons hitting the
> resistor and wiring, (increasing their effective QE) but this increases
> their capacitance (making them more susceptible to pulse-pileup).
>
> All will be well as long as the number of photons absorbed in the active
> areas during time period RC is small (5%?) compared to the number of APDs in
> the array THAT ARE ILLUMINATED BY THE BEAM.
>
> The caps above are to remind everyone that, to work properly, the size of
> the ray bundle striking the MPPC must be matched to the size of the APD
> array (1.3 to 6 mm square). This can be a problem if we imagine the ray
> bundle being limited by a confocal aperture that can be varied in size over
> a substantial range. (Do we need a zoom lens to make all possible signal ray
> -bundles match the size of the MPPC array?)
>
> Apart from this, I would like to second the comment that deep imaging is
> usually limited by spherical aberration and systems that can correct for
> this without “bumping the specimen while you try to adjust the collar” are
> to be preferred.
>
> I would also like to reaffirm that, assuming the pixel size meets Nyquist,
> you should ONLY evaluate results after deconvolving the data with an
> appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist-
> sampled image will be at least 4 (more likely 5) pixels wide, all the noise
> terms affect single pixel values. i.e., they have frequency components at
> least 4x higher than that which can represent any real structure. In scanned
> fluorescent imaging, one deconvolves more to reduce noise than to increase “
> spatial resolution” (although you can also increase resolution as long as
> you have massive amounts of signal.)
>
> Cheers,
>
> Jim Pawley
> ****************************************
> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
> Canada, V0N3A0,
> Phone [phone], email <[hidden email]<mailto:[hidden email]><mailto:[hidden email]>>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
>
>
> On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:[hidden email]><mailto:mmodel@
> KENT.EDU<http://KENT.EDU>>> 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.
> *****
>
> We have potential users who want to quantify some kind of small aggregates
> in the brain. I am afraid that deconvolution can make noise look like such
> aggregates. Perhaps collecting a noisy image twice and comparing two
> deconvolved images might help, but that seems too much work. Am I wrong?
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> On Behalf
> Of Steffen Dietzel
> Sent: Friday, May 11, 2018 10:49 AM
> To: [hidden email]<mailto:[hidden email]>
> Subject: Re: confocal detectors and deconvolution
>
> *****
> 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.
> *****
>
> Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
> there is no big difference between HyDs and GaAsP detectors.
>
> I disagree on this. In my view, the major difference is that the HyD always
> operates in photon counting mode whether, as far as I know, the PMTs (with
> or without GaAsP) create an electron cloud of which the size is determined
> by the number of photoelectrons AND statistics, and the cloud size is then
> digitized. So the output created by one photon may vary substantially
> depending on the number of electrons created on the first dynodes (which in
> turn is a statistical process). My information may be outdated and newer
> PMTs might have extra tricks, if so please correct me.
>
> Another difference is apparently the size of the photcathode. If memory
> serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs)
> creates more dark noise. I like our HyDs a lot, I appreciate having a gray
> value of "21 photons" instead of some random number. But having said this,
> at the end of the day what counts is the sensitivity of the whole system,
> and not of the detector alone. So to do this right there is no substitute
> for testing your own samples on different machines with your applications in
> mind.
>
> As for deconvolution, yes, it can create artefacts. But so does confocal
> microscopy (a point becomes an Airy pattern, not a point). And if you do it
> right the deconvolved image will be closer to the truth than the original
> image. Should you have the third edition of the handbook around, have a look
> at the preface, last paragraph.
>
> Steffen
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>
> "
>

[Craig Brideau]

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When designing an optical system for 2P (or confocal for that matter) the
scanned beam has to be pivoted around a virtual point located at the back
aperture of the objective. If this plane was relayed to the face of the
large area detector you would effectively have a descanned detection path,
loosely speaking.

Craig

On Tue, May 15, 2018 at 7:25 PM Michael Giacomelli <[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 James,
>
> That is a very interesting post.  I've read about SiPMs before, but
> did not realize they were so far as long. The need to evenly
> illuminate a large area is slightly annoying (probably not going to
> work well for non-descanned 2P), but could be designed around for a
> descanned system as you suggest.  The specs certainly look
> interesting, especially the decreased sensitivity to strong light, and
> the very low multiplicative noise.
>
> I wonder if anyone has tried one in a scanning confocal or 2p system
> and compared to conventional PMTs (e.g. h7422s or similar)?  I did a
> quick search, but didn't see many papers using them for conventional
> confocal scanning.
>
> Mike
>
> On Mon, May 14, 2018 at 10:50 PM, JAMES B 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.
> > *****
> >
> > Hi Zdenek,
> >
> > Nice slide show reference! Thanks. I will steal some images!
> >
> > However…
> >
> > I think it is a shame that they do not really discuss the difference in
> “excess noise” between PMTs and SiPMs as this can affect effective QE very
> strongly.
> >
> > Also, I think that they could have done a bit more to separate
> applications involving diffuse light from those involving almost-coherent
> light, like scanning microscopy, in which the signal originates from a very
> small volume (and therefore can be focussed onto any surface that is even
> slightly bigger). Of course, PMTs can have huge photocathodes (the R877-100
> is 12,860 mm2) and, depending on their temp and IR sensitivity, can have
> very low dark count rates per square mm of photocathode. But in scanning
> light microscopy, there seems to be little need for many square mm of
> active area.
> >
> > And even PMTs can have dark current problems. The first version of the
> Zeiss 510 used a large number (23? Don’t remember exactly.) of tiny
> servo-motors in which the windings were always excited. This heated the
> inside of the optics box enough to produce so many dark counts from their
> Hamamatsu PMT modules that they had to replace the servos with DC versions
> that were only excited when you changed some setting. And this was long
> before GaAsP photocathodes were common. From slide 19, you can see that the
> dark count rate of GaAsP is about 100x that of the more common bi-alkali
> PMTs.
> >
> > All the same, silicon diodes do leak current at room temperature and so
> it is not surprising that most MPPCs (or SiPMs) are  supplied with Peltier
> cooling systems.
> >
> > For most low-light, confocal applications it would seem that the S14420
> series might work. It has a 1.5 x 1.5 mm array containing 900, 50µm cells
> with a fill factor of 81%, a PDE of 40% and a maximum dark count of 1,000
> cps (or only 0.01 count per (long!) 10µs pixel).
> >
> > https://www.hamamatsu.com/resources/pdf/ssd/s14420_series_kapd1061e.pdf
> >
> > I know that a max PDE of 40% might sound less than the 40% QE on the
> plots of GaAsP PMT photocathodes, but these are different specs: PMT QE
> only refers to the fraction of photons making photoelectrons. About 30% of
> these photoelectrons fail to propagate down the electron-multiplier chain
> (i.e., they are lost to the signal). If you also include the fact that the
> MPPC has virtually no excess noise, this makes at least an additional 40%
> improvement in the effective QE.
> >
> > BUT you have to make sure that the ray bundle fills the array.
> >
> > (OK, maybe you don’t. Let’s assume that 600 of the 900 APDs on this
> device fall within a circle that just touches the sides of the square and
> that your signal ray-bundle just fills this circle (uniformly!) when your
> pinhole is open all the way. If we guess (from Slide 22 of the slide show)
> that the effective RC decay time is 40ns and choose a pixel time of 1 µs
> (or 25 decay times), a simple-minded analysis would lead us to conclude
> that a (massive) signal of 600 +/-24 detected photons/1 µs-pixel would lose
> close to 25 photon pulses due to pulse pile-up (i.e., a number similar the
> Poisson Noise uncertainty). A detected signal of 64 +/- 8 photons/1µs pixel
> from a darker area would lose less than one count, an error much smaller
> than the Poisson noise.
> >
> > However, if you keep the same signal levels but use a pinhole that is
> 3.3 x smaller in diameter, the signal will now hit only 60 APDs and a 600
> PE signal will lose 40% because of pulse pile-up. On the other hand, if the
> signal getting through the smaller pinhole is has gone down with the
> pinhole area and is now only 64 +/-8 pulses, then the loss is only 4%, or
> about 3 counts; smaller than the Poisson noise. For 10µs pixels, the signal
> would go up by 10 times but the percentage loss would be the same..
> >
> > So using this detector, you would have another thing to worry about: “At
> this signal level and this pinhole size, am I losing signal to pileup?”
> >
> > Annoying, but a geometric factor that the computer could easily keep
> track of and warn you when there was a problem.
> >
> > A worse problem is that, if the bundle diameter is larger than the
> back-projected image of the Airy Disk, the light in the bundle is NOT
> evenly distributed  As long as there isn’t much spherical aberration, the
> image of the spot at the pinhole is always a LOT brighter in the centre. So
> this might be a potential problem, one that could be reduced by using a
> larger array (3x3 or 6x6 mm) and changing the optics to fill these arrays.
> Larger arrays cost more and would increase dark counts by about 4x or 16x
> (resp.) but probably still not enough to worry about.
> >
> > Happy designing.
> >
> > Jim Pawley
> >
> >               ****************************************
> > James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
> BC, Canada, V0N3A0,
> > Phone [phone], email <[hidden email]<mailto:[hidden email]>>
> > NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> 1-604-989-6146
> >
> > On May 14, 18, at 9:23 AM, [hidden email]<mailto:[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<http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> >
> > To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call
> them),
> > note that their dark noise is some 3 - 4 orders of magnitude higher than
> > that of regular PMTs! Of course, chilling the detector to -80 degC
> (which is
> > common with EMCCDs, for example) would solve this issue, but the cost
> would
> > be prohibitive...
> > For more details on SiPMs vs PMTs see here:
> >
> > https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB
> >
> >
> > Cheers, zdenek
> >
> >
> >
> > ---------- Původní e-mail ----------
> > Od: JAMES B PAWLEY <[hidden email]<mailto:[hidden email]>>
> > Komu: [hidden email]<mailto:
> [hidden email]>
> > Datum: 14. 5. 2018 0:27:35
> > Předmět: Re: confocal detectors and deconvolution
> > "*****
> > 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<http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > Hi all,
> >
> > I would like to Eecho Michael’s points.
> >
> > Because hybrid photodetectors have a very high gain (>>1,000x) in their
> > first stage, they produce very little “excess noise” (also called
> > Multiplicative Noise). As a result, it is possible to characterize their
> > output as "25 photons detected” (although it might be safer to think of
> it
> > as "25 photons detected this time" or ”25 +/- 5 photons.").
> >
> > Straight PMTs do not share this feature and because single photoelectrons
> > produce output pulses that vary significantly in size, even the very best
> > PMTs produce an uncertainty in the magnitude of the signal presented to
> the
> > ADC that is at least 40% larger in relative terms than would be the case
> in
> > the absence of this excess noise. On PMTs having electron multipliers
> > optimized for other reasons (such as making them very small, like those
> in
> > the 32-PMT linear arrays), the increase in uncertainty is closer to 100%
> (i.
> > e., The signal has the same uncertainty that it would have if 4 times
> fewer
> > photons were counted perfectly.)
> >
> > Either type of PMT can have a GaAsP photocathode but it will need to be
> > cooled.
> >
> > Although single APDs may have a high photon detection efficiency (PDE, a
> > spec that is like QE but which includes the signal lost by photoelectrons
> > that do not avalanche at all) they have such massive excess noise that
> it is
> > essential to use them with pulse-counting circuits and these circuits are
> > just too slow for use in beam-scanning light microscopy.
> >
> > The solution is the multi-pixel photon counter (MPPC, a development from
> the
> > SPAD (single photon avalanche device),
> >
> > https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf
> >
> >
> https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_
> > ultimate_photosensor/index.html ).
> >
> > The surface of an MPPC is covered with an array of 400 to 20,000 APDs,
> each
> > connected to the high-voltage rail through its own damping resister. The
> > resistor causes the voltage across the APD to drop as the avalanche
> > proceeds. This quenches the discharge and produces single-photon pulses
> of
> > very uniform size. As all the APDs are electrically in parallel, these
> > single-photon current pulses simply add up producing an output current
> > signal almost devoid of excess noise. What could be better? And in
> addition
> > they are significantly less sensitive than hybrid PMTs to overheating
> damage
> > if accidentally exposed to a bright light.
> >
> > There are of course limitations: 1) A significant fraction (20-40%) of
> the
> > MPPC's surface is taken up with the resistors and the wiring to provide
> each
> > APD with + and - voltages. Photons absorbed or reflected in these areas
> are
> > lost. 2) The system is only free of pulse-pileup losses to the extent
> that
> > no APD absorbs more than one photon within its RC relaxation time (set by
> > the R of the resistor, and the capacitance (C) of the sensitive area of
> the
> > APD. Larger individual APDs “waste” proportionally fewer photons hitting
> the
> > resistor and wiring, (increasing their effective QE) but this increases
> > their capacitance (making them more susceptible to pulse-pileup).
> >
> > All will be well as long as the number of photons absorbed in the active
> > areas during time period RC is small (5%?) compared to the number of
> APDs in
> > the array THAT ARE ILLUMINATED BY THE BEAM.
> >
> > The caps above are to remind everyone that, to work properly, the size of
> > the ray bundle striking the MPPC must be matched to the size of the APD
> > array (1.3 to 6 mm square). This can be a problem if we imagine the ray
> > bundle being limited by a confocal aperture that can be varied in size
> over
> > a substantial range. (Do we need a zoom lens to make all possible signal
> ray
> > -bundles match the size of the MPPC array?)
> >
> > Apart from this, I would like to second the comment that deep imaging is
> > usually limited by spherical aberration and systems that can correct for
> > this without “bumping the specimen while you try to adjust the collar”
> are
> > to be preferred.
> >
> > I would also like to reaffirm that, assuming the pixel size meets
> Nyquist,
> > you should ONLY evaluate results after deconvolving the data with an
> > appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist-
> > sampled image will be at least 4 (more likely 5) pixels wide, all the
> noise
> > terms affect single pixel values. i.e., they have frequency components at
> > least 4x higher than that which can represent any real structure. In
> scanned
> > fluorescent imaging, one deconvolves more to reduce noise than to
> increase “
> > spatial resolution” (although you can also increase resolution as long as
> > you have massive amounts of signal.)
> >
> > Cheers,
> >
> > Jim Pawley
> > ****************************************
> > James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
> > Canada, V0N3A0,
> > Phone [phone], email <[hidden email]<mailto:[hidden email]
> ><mailto:[hidden email]>>
> > NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> 1-604-989-6146
> >
> >
> > On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:
> [hidden email]><mailto:mmodel@
> > KENT.EDU<http://KENT.EDU>>> 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.
> > *****
> >
> > We have potential users who want to quantify some kind of small
> aggregates
> > in the brain. I am afraid that deconvolution can make noise look like
> such
> > aggregates. Perhaps collecting a noisy image twice and comparing two
> > deconvolved images might help, but that seems too much work. Am I wrong?
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[hidden email]<mailto:
> [hidden email]>> On Behalf
> > Of Steffen Dietzel
> > Sent: Friday, May 11, 2018 10:49 AM
> > To: [hidden email]<mailto:
> [hidden email]>
> > Subject: Re: confocal detectors and deconvolution
> >
> > *****
> > 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.
> > *****
> >
> > Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
> > there is no big difference between HyDs and GaAsP detectors.
> >
> > I disagree on this. In my view, the major difference is that the HyD
> always
> > operates in photon counting mode whether, as far as I know, the PMTs
> (with
> > or without GaAsP) create an electron cloud of which the size is
> determined
> > by the number of photoelectrons AND statistics, and the cloud size is
> then
> > digitized. So the output created by one photon may vary substantially
> > depending on the number of electrons created on the first dynodes (which
> in
> > turn is a statistical process). My information may be outdated and newer
> > PMTs might have extra tricks, if so please correct me.
> >
> > Another difference is apparently the size of the photcathode. If memory
> > serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs)
> > creates more dark noise. I like our HyDs a lot, I appreciate having a
> gray
> > value of "21 photons" instead of some random number. But having said
> this,
> > at the end of the day what counts is the sensitivity of the whole system,
> > and not of the detector alone. So to do this right there is no substitute
> > for testing your own samples on different machines with your
> applications in
> > mind.
> >
> > As for deconvolution, yes, it can create artefacts. But so does confocal
> > microscopy (a point becomes an Airy pattern, not a point). And if you do
> it
> > right the deconvolved image will be closer to the truth than the original
> > image. Should you have the third edition of the handbook around, have a
> look
> > at the preface, last paragraph.
> >
> > Steffen
> >
> > --
> > ------------------------------------------------------------
> > Steffen Dietzel, PD Dr. rer. nat
> > Ludwig-Maximilians-Universität München
> > Biomedical Center (BMC)
> > Head of the Core Facility Bioimaging
> >
> > Großhaderner Straße 9
> > D-82152 Planegg-Martinsried
> > Germany
> >
> > http://www.bioimaging.bmc.med.uni-muenchen.de
> >
> > "
> >
>

[Tim Feinstein]

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Hi folks,

In principle I love these new GPU-enabled in-line deconvolution options.   Average users really benefit from having it "just work" almost invisibly in line with acquisition.  However, after some time using Hyvolution2 on a SP8 I have some concerns.   I should acknowledge that I am a 'practical' user and not as versed in theory as many, so I'd be grateful for any corrections.  

Most problematically, the built-in app gives almost no access to default settings.  I especially dislike the standard export option that re-scales the max intensity of each channel to a full 16 bits.  I think it's reasonable to use deconvolved data for quantitative comparisons, but you can't do that when every channel is re-scaled semi-arbitrarily.  In general I don't see the point of an output that I cannot use for quantitative comparisons.  This sets aside the question of confocal's quantitative accuracy vs. other imaging modalities, which seems like a topic for another thread.    

Someone else brought up whether deconvolving can introduce new artifacts or just highlights existing problems like spherical aberration.  Mis-modeling of the PSF can certainly make things worse, but I especially recommend keeping an eye on the signal-to-noise ratio that you enter for each channel.  Huygens problematically defaults to a near-perfect SNR of 20.  If you assign a near-perfect SNR to a noisy image, the algorithm tries to model random noise into real structures and produces a noticeable patterning artifact.  For visibly noisy images you want to set the SNR somewhere from 3 (worst case scenario) to 5 or 6 (light but visible noise).  I max out at about 10, since throughput and sample stability work against super-mega averaging and frame integration.  Since you get much more benefit from deconvolving noisy images, I'd say that the cases where decon is most useful are also the cases where it's most important to update the default settings.  

The main user option in Hyvolution is a slider that changes the resolution/image size from one that's appropriate for fast-ish imaging (~1.5x Nyquist) to a somewhat ludicrous  4-5x Nyquist.  I am concerned that most users will think they are gaining real detail at the far end of that slider.  

Fortunately it comes with a Huygens license that does batch processing, so I get my work done fast without much trouble (dios mio, GPU processing).  

For the most part you could address my concerns with a small software update.  In fact Leica may have done with their Lightning module; I have not tried it yet.  However if advanced have a hard time getting into the 'guts' of the decon algorithm then I am not sure how useful it would be.  

All the best!


Tim

Timothy Feinstein, Ph.D.
Research Scientist
Department of Developmental Biology
University of Pittsburgh

 


On 5/10/18, 10:01 AM, "Confocal Microscopy List on behalf of MODEL, MICHAEL" <[hidden email] on behalf of [hidden email]> wrote:

    *****
    To join, leave or search the confocal microscopy listserv, go to:
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    Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7C330a517696484b0637ff08d5b67e8850%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=6nt0Am7vl0aGFtMel9mh%2FdlnRkAnuJ2GIsDmZVXI9A0%3D&reserved=0 and include the link in your posting.
    *****
   
    Dear colleagues,
   
   
   
    we are looking for a new confocal microscope, and I have two questions so far.
   
    1.      Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then.
   
    2.      Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't  had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra.
   
   
   
    Thank you
   
   
   
    Mike Model
   
    Kent State University
   

[James Pawley]

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Hi Craig,

Your analysis is correct BUT only for light that proceeds in straight lines from the in-focus plane.

Non-rescanned detection is usually used with 2P microscopy to detect light that was excited at a particular time (rather than location, as this has been defined by the 2P excitation itself). Using time alone to decode location allows one to utilize signal photons that have been scattered before reaching the objective. This works optimally if you place a PMT that is totally insensitive to the 2P excitation directly under the specimen. Few systems manage this and the fallback is to collect light that enters the objective. You don’t need much optical analysis to show that light emerging from a scattering event a few tens of µm towards the objective will emerge from this lens as a rapidly diverging ray bundle that doesn’t in any way concentrate at the BFP. Any optical components (apart from a light pipe) used to try to capture any such rays will only make the situation worse.

Even large SiPM  sensors are only 6 mm on a side, considerably smaller than the BFP of a 40x NA.1.2NA objective. Consequently, as installing a "beam-splitter photodetector" immediately behind the objective is difficult, PMTs with photocathodes many mm in diameter work best for this sort of methode-non-descanned detection because they can intercept more of this rapidly-diverging beam.

Best,

Jim P.
              ****************************************
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

> On May 15, 18, at 11:06 PM, Craig Brideau <[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.
> *****
>
> When designing an optical system for 2P (or confocal for that matter) the
> scanned beam has to be pivoted around a virtual point located at the back
> aperture of the objective. If this plane was relayed to the face of the
> large area detector you would effectively have a descanned detection path,
> loosely speaking.
>
> Craig
>
> On Tue, May 15, 2018 at 7:25 PM Michael Giacomelli <[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 James,
>>
>> That is a very interesting post.  I've read about SiPMs before, but
>> did not realize they were so far as long. The need to evenly
>> illuminate a large area is slightly annoying (probably not going to
>> work well for non-descanned 2P), but could be designed around for a
>> descanned system as you suggest.  The specs certainly look
>> interesting, especially the decreased sensitivity to strong light, and
>> the very low multiplicative noise.
>>
>> I wonder if anyone has tried one in a scanning confocal or 2p system
>> and compared to conventional PMTs (e.g. h7422s or similar)?  I did a
>> quick search, but didn't see many papers using them for conventional
>> confocal scanning.
>>
>> Mike
>>
>> On Mon, May 14, 2018 at 10:50 PM, JAMES B 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.
>>> *****
>>>
>>> Hi Zdenek,
>>>
>>> Nice slide show reference! Thanks. I will steal some images!
>>>
>>> However…
>>>
>>> I think it is a shame that they do not really discuss the difference in
>> “excess noise” between PMTs and SiPMs as this can affect effective QE very
>> strongly.
>>>
>>> Also, I think that they could have done a bit more to separate
>> applications involving diffuse light from those involving almost-coherent
>> light, like scanning microscopy, in which the signal originates from a very
>> small volume (and therefore can be focussed onto any surface that is even
>> slightly bigger). Of course, PMTs can have huge photocathodes (the R877-100
>> is 12,860 mm2) and, depending on their temp and IR sensitivity, can have
>> very low dark count rates per square mm of photocathode. But in scanning
>> light microscopy, there seems to be little need for many square mm of
>> active area.
>>>
>>> And even PMTs can have dark current problems. The first version of the
>> Zeiss 510 used a large number (23? Don’t remember exactly.) of tiny
>> servo-motors in which the windings were always excited. This heated the
>> inside of the optics box enough to produce so many dark counts from their
>> Hamamatsu PMT modules that they had to replace the servos with DC versions
>> that were only excited when you changed some setting. And this was long
>> before GaAsP photocathodes were common. From slide 19, you can see that the
>> dark count rate of GaAsP is about 100x that of the more common bi-alkali
>> PMTs.
>>>
>>> All the same, silicon diodes do leak current at room temperature and so
>> it is not surprising that most MPPCs (or SiPMs) are  supplied with Peltier
>> cooling systems.
>>>
>>> For most low-light, confocal applications it would seem that the S14420
>> series might work. It has a 1.5 x 1.5 mm array containing 900, 50µm cells
>> with a fill factor of 81%, a PDE of 40% and a maximum dark count of 1,000
>> cps (or only 0.01 count per (long!) 10µs pixel).
>>>
>>> https://www.hamamatsu.com/resources/pdf/ssd/s14420_series_kapd1061e.pdf
>>>
>>> I know that a max PDE of 40% might sound less than the 40% QE on the
>> plots of GaAsP PMT photocathodes, but these are different specs: PMT QE
>> only refers to the fraction of photons making photoelectrons. About 30% of
>> these photoelectrons fail to propagate down the electron-multiplier chain
>> (i.e., they are lost to the signal). If you also include the fact that the
>> MPPC has virtually no excess noise, this makes at least an additional 40%
>> improvement in the effective QE.
>>>
>>> BUT you have to make sure that the ray bundle fills the array.
>>>
>>> (OK, maybe you don’t. Let’s assume that 600 of the 900 APDs on this
>> device fall within a circle that just touches the sides of the square and
>> that your signal ray-bundle just fills this circle (uniformly!) when your
>> pinhole is open all the way. If we guess (from Slide 22 of the slide show)
>> that the effective RC decay time is 40ns and choose a pixel time of 1 µs
>> (or 25 decay times), a simple-minded analysis would lead us to conclude
>> that a (massive) signal of 600 +/-24 detected photons/1 µs-pixel would lose
>> close to 25 photon pulses due to pulse pile-up (i.e., a number similar the
>> Poisson Noise uncertainty). A detected signal of 64 +/- 8 photons/1µs pixel
>> from a darker area would lose less than one count, an error much smaller
>> than the Poisson noise.
>>>
>>> However, if you keep the same signal levels but use a pinhole that is
>> 3.3 x smaller in diameter, the signal will now hit only 60 APDs and a 600
>> PE signal will lose 40% because of pulse pile-up. On the other hand, if the
>> signal getting through the smaller pinhole is has gone down with the
>> pinhole area and is now only 64 +/-8 pulses, then the loss is only 4%, or
>> about 3 counts; smaller than the Poisson noise. For 10µs pixels, the signal
>> would go up by 10 times but the percentage loss would be the same..
>>>
>>> So using this detector, you would have another thing to worry about: “At
>> this signal level and this pinhole size, am I losing signal to pileup?”
>>>
>>> Annoying, but a geometric factor that the computer could easily keep
>> track of and warn you when there was a problem.
>>>
>>> A worse problem is that, if the bundle diameter is larger than the
>> back-projected image of the Airy Disk, the light in the bundle is NOT
>> evenly distributed  As long as there isn’t much spherical aberration, the
>> image of the spot at the pinhole is always a LOT brighter in the centre. So
>> this might be a potential problem, one that could be reduced by using a
>> larger array (3x3 or 6x6 mm) and changing the optics to fill these arrays.
>> Larger arrays cost more and would increase dark counts by about 4x or 16x
>> (resp.) but probably still not enough to worry about.
>>>
>>> Happy designing.
>>>
>>> Jim Pawley
>>>
>>>              ****************************************
>>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
>> BC, Canada, V0N3A0,
>>> Phone [phone], email <[hidden email]<mailto:[hidden email]>>
>>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
>> 1-604-989-6146
>>>
>>> On May 14, 18, at 9:23 AM, [hidden email]<mailto:[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<http://www.imgur.com/> and include
>> the link in your posting.
>>> *****
>>>
>>>
>>> To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call
>> them),
>>> note that their dark noise is some 3 - 4 orders of magnitude higher than
>>> that of regular PMTs! Of course, chilling the detector to -80 degC
>> (which is
>>> common with EMCCDs, for example) would solve this issue, but the cost
>> would
>>> be prohibitive...
>>> For more details on SiPMs vs PMTs see here:
>>>
>>> https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB
>>>
>>>
>>> Cheers, zdenek
>>>
>>>
>>>
>>> ---------- Původní e-mail ----------
>>> Od: JAMES B PAWLEY <[hidden email]<mailto:[hidden email]>>
>>> Komu: [hidden email]<mailto:
>> [hidden email]>
>>> Datum: 14. 5. 2018 0:27:35
>>> Předmět: Re: confocal detectors and deconvolution
>>> "*****
>>> 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<http://www.imgur.com/> and include
>> the link in your posting.
>>> *****
>>>
>>> Hi all,
>>>
>>> I would like to Eecho Michael’s points.
>>>
>>> Because hybrid photodetectors have a very high gain (>>1,000x) in their
>>> first stage, they produce very little “excess noise” (also called
>>> Multiplicative Noise). As a result, it is possible to characterize their
>>> output as "25 photons detected” (although it might be safer to think of
>> it
>>> as "25 photons detected this time" or ”25 +/- 5 photons.").
>>>
>>> Straight PMTs do not share this feature and because single photoelectrons
>>> produce output pulses that vary significantly in size, even the very best
>>> PMTs produce an uncertainty in the magnitude of the signal presented to
>> the
>>> ADC that is at least 40% larger in relative terms than would be the case
>> in
>>> the absence of this excess noise. On PMTs having electron multipliers
>>> optimized for other reasons (such as making them very small, like those
>> in
>>> the 32-PMT linear arrays), the increase in uncertainty is closer to 100%
>> (i.
>>> e., The signal has the same uncertainty that it would have if 4 times
>> fewer
>>> photons were counted perfectly.)
>>>
>>> Either type of PMT can have a GaAsP photocathode but it will need to be
>>> cooled.
>>>
>>> Although single APDs may have a high photon detection efficiency (PDE, a
>>> spec that is like QE but which includes the signal lost by photoelectrons
>>> that do not avalanche at all) they have such massive excess noise that
>> it is
>>> essential to use them with pulse-counting circuits and these circuits are
>>> just too slow for use in beam-scanning light microscopy.
>>>
>>> The solution is the multi-pixel photon counter (MPPC, a development from
>> the
>>> SPAD (single photon avalanche device),
>>>
>>> https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf
>>>
>>>
>> https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_
>>> ultimate_photosensor/index.html ).
>>>
>>> The surface of an MPPC is covered with an array of 400 to 20,000 APDs,
>> each
>>> connected to the high-voltage rail through its own damping resister. The
>>> resistor causes the voltage across the APD to drop as the avalanche
>>> proceeds. This quenches the discharge and produces single-photon pulses
>> of
>>> very uniform size. As all the APDs are electrically in parallel, these
>>> single-photon current pulses simply add up producing an output current
>>> signal almost devoid of excess noise. What could be better? And in
>> addition
>>> they are significantly less sensitive than hybrid PMTs to overheating
>> damage
>>> if accidentally exposed to a bright light.
>>>
>>> There are of course limitations: 1) A significant fraction (20-40%) of
>> the
>>> MPPC's surface is taken up with the resistors and the wiring to provide
>> each
>>> APD with + and - voltages. Photons absorbed or reflected in these areas
>> are
>>> lost. 2) The system is only free of pulse-pileup losses to the extent
>> that
>>> no APD absorbs more than one photon within its RC relaxation time (set by
>>> the R of the resistor, and the capacitance (C) of the sensitive area of
>> the
>>> APD. Larger individual APDs “waste” proportionally fewer photons hitting
>> the
>>> resistor and wiring, (increasing their effective QE) but this increases
>>> their capacitance (making them more susceptible to pulse-pileup).
>>>
>>> All will be well as long as the number of photons absorbed in the active
>>> areas during time period RC is small (5%?) compared to the number of
>> APDs in
>>> the array THAT ARE ILLUMINATED BY THE BEAM.
>>>
>>> The caps above are to remind everyone that, to work properly, the size of
>>> the ray bundle striking the MPPC must be matched to the size of the APD
>>> array (1.3 to 6 mm square). This can be a problem if we imagine the ray
>>> bundle being limited by a confocal aperture that can be varied in size
>> over
>>> a substantial range. (Do we need a zoom lens to make all possible signal
>> ray
>>> -bundles match the size of the MPPC array?)
>>>
>>> Apart from this, I would like to second the comment that deep imaging is
>>> usually limited by spherical aberration and systems that can correct for
>>> this without “bumping the specimen while you try to adjust the collar”
>> are
>>> to be preferred.
>>>
>>> I would also like to reaffirm that, assuming the pixel size meets
>> Nyquist,
>>> you should ONLY evaluate results after deconvolving the data with an
>>> appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist-
>>> sampled image will be at least 4 (more likely 5) pixels wide, all the
>> noise
>>> terms affect single pixel values. i.e., they have frequency components at
>>> least 4x higher than that which can represent any real structure. In
>> scanned
>>> fluorescent imaging, one deconvolves more to reduce noise than to
>> increase “
>>> spatial resolution” (although you can also increase resolution as long as
>>> you have massive amounts of signal.)
>>>
>>> Cheers,
>>>
>>> Jim Pawley
>>> ****************************************
>>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
>>> Canada, V0N3A0,
>>> Phone [phone], email <[hidden email]<mailto:[hidden email]
>>> <mailto:[hidden email]>>
>>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
>> 1-604-989-6146
>>>
>>>
>>> On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:
>> [hidden email]><mailto:mmodel@
>>> KENT.EDU<http://KENT.EDU>>> 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.
>>> *****
>>>
>>> We have potential users who want to quantify some kind of small
>> aggregates
>>> in the brain. I am afraid that deconvolution can make noise look like
>> such
>>> aggregates. Perhaps collecting a noisy image twice and comparing two
>>> deconvolved images might help, but that seems too much work. Am I wrong?
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List <[hidden email]<mailto:
>> [hidden email]>> On Behalf
>>> Of Steffen Dietzel
>>> Sent: Friday, May 11, 2018 10:49 AM
>>> To: [hidden email]<mailto:
>> [hidden email]>
>>> Subject: Re: confocal detectors and deconvolution
>>>
>>> *****
>>> 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.
>>> *****
>>>
>>> Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
>>> there is no big difference between HyDs and GaAsP detectors.
>>>
>>> I disagree on this. In my view, the major difference is that the HyD
>> always
>>> operates in photon counting mode whether, as far as I know, the PMTs
>> (with
>>> or without GaAsP) create an electron cloud of which the size is
>> determined
>>> by the number of photoelectrons AND statistics, and the cloud size is
>> then
>>> digitized. So the output created by one photon may vary substantially
>>> depending on the number of electrons created on the first dynodes (which
>> in
>>> turn is a statistical process). My information may be outdated and newer
>>> PMTs might have extra tricks, if so please correct me.
>>>
>>> Another difference is apparently the size of the photcathode. If memory
>>> serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs)
>>> creates more dark noise. I like our HyDs a lot, I appreciate having a
>> gray
>>> value of "21 photons" instead of some random number. But having said
>> this,
>>> at the end of the day what counts is the sensitivity of the whole system,
>>> and not of the detector alone. So to do this right there is no substitute
>>> for testing your own samples on different machines with your
>> applications in
>>> mind.
>>>
>>> As for deconvolution, yes, it can create artefacts. But so does confocal
>>> microscopy (a point becomes an Airy pattern, not a point). And if you do
>> it
>>> right the deconvolved image will be closer to the truth than the original
>>> image. Should you have the third edition of the handbook around, have a
>> look
>>> at the preface, last paragraph.
>>>
>>> Steffen
>>>
>>> --
>>> ------------------------------------------------------------
>>> Steffen Dietzel, PD Dr. rer. nat
>>> Ludwig-Maximilians-Universität München
>>> Biomedical Center (BMC)
>>> Head of the Core Facility Bioimaging
>>>
>>> Großhaderner Straße 9
>>> D-82152 Planegg-Martinsried
>>> Germany
>>>
>>> http://www.bioimaging.bmc.med.uni-muenchen.de
>>>
>>> "
>>>
>>

[George McNamara]

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Hi Tim,

Now that Leica is divorced from SVI on HyVolution2, maybe SVI will
provide an update to its software that can ignore - at your choice - the
settings LAS X pushes over with the data.

I also suggest that small (200 nm?) tetraspeck beads, or better
refractive index matched 'objects', sparsely on the coverglass, and
optionally on the 'back side' surface (slide if
coverglass-specimen-slide prep), or very sparse in the (viscous to make
the 'beads' immobile) mounting medium (i.e. Prolong Glass, Opti-Bryt
when using conventional oil immersion lenses; Iodixanol= OptiPrep :
aqueous cultuere=mounting medium) would be "just a little brighter" than
the brightest specimen voxels, to get HyVolution2 to behave better (ok,
simpler for SVI to improve Huygens to let us override stupid Leica shit).


enjoy,

George


On 5/16/2018 11:05 AM, Feinstein, Timothy N 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 folks,
>
> In principle I love these new GPU-enabled in-line deconvolution options.   Average users really benefit from having it "just work" almost invisibly in line with acquisition.  However, after some time using Hyvolution2 on a SP8 I have some concerns.   I should acknowledge that I am a 'practical' user and not as versed in theory as many, so I'd be grateful for any corrections.
>
> Most problematically, the built-in app gives almost no access to default settings.  I especially dislike the standard export option that re-scales the max intensity of each channel to a full 16 bits.  I think it's reasonable to use deconvolved data for quantitative comparisons, but you can't do that when every channel is re-scaled semi-arbitrarily.  In general I don't see the point of an output that I cannot use for quantitative comparisons.  This sets aside the question of confocal's quantitative accuracy vs. other imaging modalities, which seems like a topic for another thread.
>
> Someone else brought up whether deconvolving can introduce new artifacts or just highlights existing problems like spherical aberration.  Mis-modeling of the PSF can certainly make things worse, but I especially recommend keeping an eye on the signal-to-noise ratio that you enter for each channel.  Huygens problematically defaults to a near-perfect SNR of 20.  If you assign a near-perfect SNR to a noisy image, the algorithm tries to model random noise into real structures and produces a noticeable patterning artifact.  For visibly noisy images you want to set the SNR somewhere from 3 (worst case scenario) to 5 or 6 (light but visible noise).  I max out at about 10, since throughput and sample stability work against super-mega averaging and frame integration.  Since you get much more benefit from deconvolving noisy images, I'd say that the cases where decon is most useful are also the cases where it's most important to update the default settings.
>
> The main user option in Hyvolution is a slider that changes the resolution/image size from one that's appropriate for fast-ish imaging (~1.5x Nyquist) to a somewhat ludicrous  4-5x Nyquist.  I am concerned that most users will think they are gaining real detail at the far end of that slider.
>
> Fortunately it comes with a Huygens license that does batch processing, so I get my work done fast without much trouble (dios mio, GPU processing).
>
> For the most part you could address my concerns with a small software update.  In fact Leica may have done with their Lightning module; I have not tried it yet.  However if advanced have a hard time getting into the 'guts' of the decon algorithm then I am not sure how useful it would be.
>
> All the best!
>
>
> Tim
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> Department of Developmental Biology
> University of Pittsburgh
>
>  
>
>
> On 5/10/18, 10:01 AM, "Confocal Microscopy List on behalf of MODEL, MICHAEL" <[hidden email] on behalf of [hidden email]> wrote:
>
>      *****
>      To join, leave or search the confocal microscopy listserv, go to:
>      https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Ctnf8%40PITT.EDU%7C330a517696484b0637ff08d5b67e8850%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=DblPqLXZqUdkV4psnBLFi8fNsOQXpUFJr%2BwqPhoS7YQ%3D&reserved=0
>      Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7C330a517696484b0637ff08d5b67e8850%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=6nt0Am7vl0aGFtMel9mh%2FdlnRkAnuJ2GIsDmZVXI9A0%3D&reserved=0 and include the link in your posting.
>      *****
>      
>      Dear colleagues,
>      
>      
>      
>      we are looking for a new confocal microscope, and I have two questions so far.
>      
>      1.      Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then.
>      
>      2.      Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't  had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra.
>      
>      
>      
>      Thank you
>      
>      
>      
>      Mike Model
>      
>      Kent State University
>      
>

--


George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures

[Avi Jacob]

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I always set the slider such that the pinhole goes down to 80%, and that is
what I teach the users. The slider has no units itself, so one cannot know
if you are working with comparable settings between sets of images, or, as
you say, you can easily get into unrealistic territory. But because
everything else is derived from the pinhole (I think), that is a good way,
IMO, to "normalize" settings. Theoretically, going down more, should
improve the resolution even more, yet, as we know, it all a matter of
balance.
I also frequently unclick the padlock and change some of the parameters.
Looking forward to seeing if they allowed decon parameter changes in
Lightning. I'll be at elmi in Dublin, hope to meet some of you there.
Avi





On Wed, May 16, 2018 at 6:05 PM, Feinstein, Timothy N <[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 folks,
>
> In principle I love these new GPU-enabled in-line deconvolution options.
>  Average users really benefit from having it "just work" almost invisibly
> in line with acquisition.  However, after some time using Hyvolution2 on a
> SP8 I have some concerns.   I should acknowledge that I am a 'practical'
> user and not as versed in theory as many, so I'd be grateful for any
> corrections.
>
> Most problematically, the built-in app gives almost no access to default
> settings.  I especially dislike the standard export option that re-scales
> the max intensity of each channel to a full 16 bits.  I think it's
> reasonable to use deconvolved data for quantitative comparisons, but you
> can't do that when every channel is re-scaled semi-arbitrarily.  In general
> I don't see the point of an output that I cannot use for quantitative
> comparisons.  This sets aside the question of confocal's quantitative
> accuracy vs. other imaging modalities, which seems like a topic for another
> thread.
>
> Someone else brought up whether deconvolving can introduce new artifacts
> or just highlights existing problems like spherical aberration.
> Mis-modeling of the PSF can certainly make things worse, but I especially
> recommend keeping an eye on the signal-to-noise ratio that you enter for
> each channel.  Huygens problematically defaults to a near-perfect SNR of
> 20.  If you assign a near-perfect SNR to a noisy image, the algorithm tries
> to model random noise into real structures and produces a noticeable
> patterning artifact.  For visibly noisy images you want to set the SNR
> somewhere from 3 (worst case scenario) to 5 or 6 (light but visible
> noise).  I max out at about 10, since throughput and sample stability work
> against super-mega averaging and frame integration.  Since you get much
> more benefit from deconvolving noisy images, I'd say that the cases where
> decon is most useful are also the cases where it's most important to update
> the default settings.
>
> The main user option in Hyvolution is a slider that changes the
> resolution/image size from one that's appropriate for fast-ish imaging
> (~1.5x Nyquist) to a somewhat ludicrous  4-5x Nyquist.  I am concerned that
> most users will think they are gaining real detail at the far end of that
> slider.
>
> Fortunately it comes with a Huygens license that does batch processing, so
> I get my work done fast without much trouble (dios mio, GPU processing).
>
> For the most part you could address my concerns with a small software
> update.  In fact Leica may have done with their Lightning module; I have
> not tried it yet.  However if advanced have a hard time getting into the
> 'guts' of the decon algorithm then I am not sure how useful it would be.
>
> All the best!
>
>
> Tim
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> Department of Developmental Biology
> University of Pittsburgh
>
>
>

[0000001ed7f52e4a-dmarc-request]

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Hello,
 
I find the SNR parameter default of 20 actually quite good (using Huygens standalone software). I found that the result is actually less sensitive to changes in this parameter when I use the Nyquist sampled pixel size from the SVI Nyquist calculator, e.g. 43 nm for NA 1.4 oil immersion. When using 'more sensible' larger pixel sizes, changing this parameter easily introduces artefacts. Now Huygens has a preview to easily see the result before deconvolving the whole stack. I think pinhole should be adjusted independent from other parameters.

best wishes

Andreas
 
 
-----Original Message-----
From: Feinstein, Timothy N <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Wed, 16 May 2018 16:09
Subject: Re: confocal detectors and deconvolution

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*****

Hi folks,

In principle I love these new GPU-enabled in-line deconvolution options.   Average users really benefit from having it "just work" almost invisibly in line with acquisition.  However, after some time using Hyvolution2 on a SP8 I have some concerns.   I should acknowledge that I am a 'practical' user and not as versed in theory as many, so I'd be grateful for any corrections.  

Most problematically, the built-in app gives almost no access to default settings.  I especially dislike the standard export option that re-scales the max intensity of each channel to a full 16 bits.  I think it's reasonable to use deconvolved data for quantitative comparisons, but you can't do that when every channel is re-scaled semi-arbitrarily.  In general I don't see the point of an output that I cannot use for quantitative comparisons.  This sets aside the question of confocal's quantitative accuracy vs. other imaging modalities, which seems like a topic for another thread.    

Someone else brought up whether deconvolving can introduce new artifacts or just highlights existing problems like spherical aberration.  Mis-modeling of the PSF can certainly make things worse, but I especially recommend keeping an eye on the signal-to-noise ratio that you enter for each channel.  Huygens problematically defaults to a near-perfect SNR of 20. If you assign a near-perfect SNR to a noisy image, the algorithm tries to model random noise into real structures and produces a noticeable patterning artifact. For visibly noisy images you want to set the SNR somewhere from 3 (worst case scenario) to 5 or 6 (light but visible noise).  I max out at about 10, since throughput and sample stability work against super-mega averaging and frame integration.  Since you get much more benefit from deconvolving noisy images, I'd say that the cases where decon is most useful are also the cases where it's most important to update the default settings.  

The main user option in Hyvolution is a slider that changes the resolution/image size from one that's appropriate for fast-ish imaging (~1.5x Nyquist) to a somewhat ludicrous  4-5x Nyquist.  I am concerned that most users will think they are gaining real detail at the far end of that slider.  

Fortunately it comes with a Huygens license that does batch processing, so I get my work done fast without much trouble (dios mio, GPU processing).  

For the most part you could address my concerns with a small software update.  In fact Leica may have done with their Lightning module; I have not tried it yet.  However if advanced have a hard time getting into the 'guts' of the decon algorithm then I am not sure how useful it would be.  

All the best!


Tim

Timothy Feinstein, Ph.D.
Research Scientist
Department of Developmental Biology
University of Pittsburgh

 


On 5/10/18, 10:01 AM, "Confocal Microscopy List on behalf of MODEL, MICHAEL" <[hidden email] on behalf of [hidden email]> wrote:

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    *****
   
    Dear colleagues,
   
   
   
    we are looking for a new confocal microscope, and I have two questions so far.
   
    1.      Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then.
   
 2.      Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't  had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra.
   
   
   
    Thank you
   
   
   
    Mike Model
   
    Kent State University
   

[Zdenek Svindrych-2]

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Hi James,
the way I couple detectors to the objective for non-descanned 2P imaging is
by relaying the BFP of the objective lens to the active area of the
detector. I do it because (1) you couple (almost) all the light that the
objective lens is able to collect, and (2) the illumination of the detector
is quite homogeneous (the sample itself is perfectly blurred on the
detector).

With your lens (40x / 1.2 NA) as an example (assuming Oil, Zeiss) with BFP
diameter around 10 mm, and Hamamatsu hybrid detector with 3 mm diameter
active area, a simple telecentric relay (f1 = 156 mm, f2 = 50 mm) will do
the trick. The only question is, how big the two lenses should be? This of
course depends on the required field of view, and it turns out to be
(paraxial approcxiamtion, 22 mm field number) 32 mm and 25 mm diameters of 
the two relay lenses. Now accounting for the defoucus of several tens of µm
due to light scattering, the relay lenses need to be bigger, but not
dramatically. For defocus of +/-1% of the objective focal length the lenses
grow to 36 mm and 29 mm diameter. Possibly less with more advanced design.


Of course to couple ALL the light that can possibly pass through the
objective, even bigger relay lenses, or different approach, would be needed,
but that depends on the particularities of each individual objective lens...

Best, zdenek
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
email: [hidden email]

---------- Původní e-mail ----------
Od: JAMES B PAWLEY <[hidden email]>
Komu: [hidden email]
Datum: 17. 5. 2018 0:42:27
Předmět: Re: confocal detectors and deconvolution
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Hi Craig,

Your analysis is correct BUT only for light that proceeds in straight lines
from the in-focus plane.

Non-rescanned detection is usually used with 2P microscopy to detect light
that was excited at a particular time (rather than location, as this has
been defined by the 2P excitation itself). Using time alone to decode
location allows one to utilize signal photons that have been scattered
before reaching the objective. This works optimally if you place a PMT that
is totally insensitive to the 2P excitation directly under the specimen. Few
systems manage this and the fallback is to collect light that enters the
objective. You don’t need much optical analysis to show that light emerging
from a scattering event a few tens of µm towards the objective will emerge
from this lens as a rapidly diverging ray bundle that doesn’t in any way
concentrate at the BFP. Any optical components (apart from a light pipe)
used to try to capture any such rays will only make the situation worse.

Even large SiPM sensors are only 6 mm on a side, considerably smaller than
the BFP of a 40x NA.1.2NA objective. Consequently, as installing a "beam-
splitter photodetector" immediately behind the objective is difficult, PMTs
with photocathodes many mm in diameter work best for this sort of methode-
non-descanned detection because they can intercept more of this rapidly-
diverging beam.

Best,

Jim P.
****************************************
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
Canada, V0N3A0,
Phone [phone], email <[hidden email]>
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> On May 15, 18, at 11:06 PM, Craig Brideau <[hidden email]> wrote:

>
> *****
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> *****
>
> When designing an optical system for 2P (or confocal for that matter) the
> scanned beam has to be pivoted around a virtual point located at the back
> aperture of the objective. If this plane was relayed to the face of the
> large area detector you would effectively have a descanned detection path,

> loosely speaking.
>
> Craig
>
> On Tue, May 15, 2018 at 7:25 PM Michael Giacomelli <[hidden email]> wrote:
>
>> *****
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>> Post images on http://www.imgur.com and include the link in your posting.

>> *****
>>
>> Hi James,
>>
>> That is a very interesting post. I've read about SiPMs before, but
>> did not realize they were so far as long. The need to evenly
>> illuminate a large area is slightly annoying (probably not going to
>> work well for non-descanned 2P), but could be designed around for a
>> descanned system as you suggest. The specs certainly look
>> interesting, especially the decreased sensitivity to strong light, and
>> the very low multiplicative noise.
>>
>> I wonder if anyone has tried one in a scanning confocal or 2p system
>> and compared to conventional PMTs (e.g. h7422s or similar)? I did a
>> quick search, but didn't see many papers using them for conventional
>> confocal scanning.
>>
>> Mike
>>
>> On Mon, May 14, 2018 at 10:50 PM, JAMES B 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.
>>> *****
>>>
>>> Hi Zdenek,
>>>
>>> Nice slide show reference! Thanks. I will steal some images!
>>>
>>> However…
>>>
>>> I think it is a shame that they do not really discuss the difference in
>> “excess noise” between PMTs and SiPMs as this can affect effective QE

very
>> strongly.
>>>
>>> Also, I think that they could have done a bit more to separate
>> applications involving diffuse light from those involving almost-coherent

>> light, like scanning microscopy, in which the signal originates from a
very
>> small volume (and therefore can be focussed onto any surface that is even

>> slightly bigger). Of course, PMTs can have huge photocathodes (the R877-
100
>> is 12,860 mm2) and, depending on their temp and IR sensitivity, can have
>> very low dark count rates per square mm of photocathode. But in scanning
>> light microscopy, there seems to be little need for many square mm of
>> active area.
>>>
>>> And even PMTs can have dark current problems. The first version of the
>> Zeiss 510 used a large number (23? Don’t remember exactly.) of tiny
>> servo-motors in which the windings were always excited. This heated the
>> inside of the optics box enough to produce so many dark counts from their

>> Hamamatsu PMT modules that they had to replace the servos with DC
versions
>> that were only excited when you changed some setting. And this was long
>> before GaAsP photocathodes were common. From slide 19, you can see that
the
>> dark count rate of GaAsP is about 100x that of the more common bi-alkali
>> PMTs.
>>>
>>> All the same, silicon diodes do leak current at room temperature and so
>> it is not surprising that most MPPCs (or SiPMs) are supplied with Peltier

>> cooling systems.
>>>
>>> For most low-light, confocal applications it would seem that the S14420
>> series might work. It has a 1.5 x 1.5 mm array containing 900, 50µm cells

>> with a fill factor of 81%, a PDE of 40% and a maximum dark count of 1,000

>> cps (or only 0.01 count per (long!) 10µs pixel).
>>>
>>> https://www.hamamatsu.com/resources/pdf/ssd/s14420_series_kapd1061e.pdf
>>>
>>> I know that a max PDE of 40% might sound less than the 40% QE on the
>> plots of GaAsP PMT photocathodes, but these are different specs: PMT QE
>> only refers to the fraction of photons making photoelectrons. About 30%
of
>> these photoelectrons fail to propagate down the electron-multiplier chain

>> (i.e., they are lost to the signal). If you also include the fact that
the
>> MPPC has virtually no excess noise, this makes at least an additional 40%

>> improvement in the effective QE.
>>>
>>> BUT you have to make sure that the ray bundle fills the array.
>>>
>>> (OK, maybe you don’t. Let’s assume that 600 of the 900 APDs on this
>> device fall within a circle that just touches the sides of the square and

>> that your signal ray-bundle just fills this circle (uniformly!) when your

>> pinhole is open all the way. If we guess (from Slide 22 of the slide
show)
>> that the effective RC decay time is 40ns and choose a pixel time of 1 µs
>> (or 25 decay times), a simple-minded analysis would lead us to conclude
>> that a (massive) signal of 600 +/-24 detected photons/1 µs-pixel would
lose
>> close to 25 photon pulses due to pulse pile-up (i.e., a number similar
the
>> Poisson Noise uncertainty). A detected signal of 64 +/- 8 photons/1µs
pixel
>> from a darker area would lose less than one count, an error much smaller
>> than the Poisson noise.
>>>
>>> However, if you keep the same signal levels but use a pinhole that is
>> 3.3 x smaller in diameter, the signal will now hit only 60 APDs and a 600

>> PE signal will lose 40% because of pulse pile-up. On the other hand, if
the
>> signal getting through the smaller pinhole is has gone down with the
>> pinhole area and is now only 64 +/-8 pulses, then the loss is only 4%, or

>> about 3 counts; smaller than the Poisson noise. For 10µs pixels, the
signal
>> would go up by 10 times but the percentage loss would be the same..
>>>
>>> So using this detector, you would have another thing to worry about: “At

>> this signal level and this pinhole size, am I losing signal to pileup?”
>>>
>>> Annoying, but a geometric factor that the computer could easily keep
>> track of and warn you when there was a problem.
>>>
>>> A worse problem is that, if the bundle diameter is larger than the
>> back-projected image of the Airy Disk, the light in the bundle is NOT
>> evenly distributed As long as there isn’t much spherical aberration, the
>> image of the spot at the pinhole is always a LOT brighter in the centre.
So
>> this might be a potential problem, one that could be reduced by using a
>> larger array (3x3 or 6x6 mm) and changing the optics to fill these
arrays.
>> Larger arrays cost more and would increase dark counts by about 4x or 16x

>> (resp.) but probably still not enough to worry about.
>>>
>>> Happy designing.
>>>
>>> Jim Pawley
>>>
>>> ****************************************
>>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
>> BC, Canada, V0N3A0,
>>> Phone [phone], email <[hidden email]<mailto:[hidden email]>>
>>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
>> 1-604-989-6146
>>>
>>> On May 14, 18, at 9:23 AM, [hidden email]<mailto:[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<http://www.imgur.com/> and include
>> the link in your posting.
>>> *****
>>>
>>>
>>> To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call
>> them),
>>> note that their dark noise is some 3 - 4 orders of magnitude higher than

>>> that of regular PMTs! Of course, chilling the detector to -80 degC
>> (which is
>>> common with EMCCDs, for example) would solve this issue, but the cost
>> would
>>> be prohibitive...
>>> For more details on SiPMs vs PMTs see here:
>>>
>>> https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB 
>>>
>>>
>>> Cheers, zdenek
>>>
>>>
>>>
>>> ---------- Původní e-mail ----------
>>> Od: JAMES B PAWLEY <[hidden email]<mailto:[hidden email]>>
>>> Komu: [hidden email]<mailto:
>> [hidden email]>
>>> Datum: 14. 5. 2018 0:27:35
>>> Předmět: Re: confocal detectors and deconvolution
>>> "*****
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>> the link in your posting.
>>> *****
>>>
>>> Hi all,
>>>
>>> I would like to Eecho Michael’s points.
>>>
>>> Because hybrid photodetectors have a very high gain (>>1,000x) in their
>>> first stage, they produce very little “excess noise” (also called
>>> Multiplicative Noise). As a result, it is possible to characterize their

>>> output as "25 photons detected” (although it might be safer to think of
>> it
>>> as "25 photons detected this time" or ”25 +/- 5 photons.").
>>>
>>> Straight PMTs do not share this feature and because single
photoelectrons
>>> produce output pulses that vary significantly in size, even the very
best
>>> PMTs produce an uncertainty in the magnitude of the signal presented to
>> the
>>> ADC that is at least 40% larger in relative terms than would be the case

>> in
>>> the absence of this excess noise. On PMTs having electron multipliers
>>> optimized for other reasons (such as making them very small, like those
>> in
>>> the 32-PMT linear arrays), the increase in uncertainty is closer to 100%

>> (i.
>>> e., The signal has the same uncertainty that it would have if 4 times
>> fewer
>>> photons were counted perfectly.)
>>>
>>> Either type of PMT can have a GaAsP photocathode but it will need to be
>>> cooled.
>>>
>>> Although single APDs may have a high photon detection efficiency (PDE, a

>>> spec that is like QE but which includes the signal lost by
photoelectrons
>>> that do not avalanche at all) they have such massive excess noise that
>> it is
>>> essential to use them with pulse-counting circuits and these circuits
are
>>> just too slow for use in beam-scanning light microscopy.
>>>
>>> The solution is the multi-pixel photon counter (MPPC, a development from

>> the
>>> SPAD (single photon avalanche device),
>>>
>>> https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf
>>>
>>>
>> https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_
the_
>>> ultimate_photosensor/index.html ).
>>>
>>> The surface of an MPPC is covered with an array of 400 to 20,000 APDs,
>> each
>>> connected to the high-voltage rail through its own damping resister. The

>>> resistor causes the voltage across the APD to drop as the avalanche
>>> proceeds. This quenches the discharge and produces single-photon pulses
>> of
>>> very uniform size. As all the APDs are electrically in parallel, these
>>> single-photon current pulses simply add up producing an output current
>>> signal almost devoid of excess noise. What could be better? And in
>> addition
>>> they are significantly less sensitive than hybrid PMTs to overheating
>> damage
>>> if accidentally exposed to a bright light.
>>>
>>> There are of course limitations: 1) A significant fraction (20-40%) of
>> the
>>> MPPC's surface is taken up with the resistors and the wiring to provide
>> each
>>> APD with + and - voltages. Photons absorbed or reflected in these areas
>> are
>>> lost. 2) The system is only free of pulse-pileup losses to the extent
>> that
>>> no APD absorbs more than one photon within its RC relaxation time (set

by
>>> the R of the resistor, and the capacitance (C) of the sensitive area of
>> the
>>> APD. Larger individual APDs “waste” proportionally fewer photons hitting

>> the
>>> resistor and wiring, (increasing their effective QE) but this increases
>>> their capacitance (making them more susceptible to pulse-pileup).
>>>
>>> All will be well as long as the number of photons absorbed in the active

>>> areas during time period RC is small (5%?) compared to the number of
>> APDs in
>>> the array THAT ARE ILLUMINATED BY THE BEAM.
>>>
>>> The caps above are to remind everyone that, to work properly, the size
of
>>> the ray bundle striking the MPPC must be matched to the size of the APD
>>> array (1.3 to 6 mm square). This can be a problem if we imagine the ray
>>> bundle being limited by a confocal aperture that can be varied in size
>> over
>>> a substantial range. (Do we need a zoom lens to make all possible signal

>> ray
>>> -bundles match the size of the MPPC array?)
>>>
>>> Apart from this, I would like to second the comment that deep imaging is

>>> usually limited by spherical aberration and systems that can correct for

>>> this without “bumping the specimen while you try to adjust the collar”
>> are
>>> to be preferred.
>>>
>>> I would also like to reaffirm that, assuming the pixel size meets
>> Nyquist,
>>> you should ONLY evaluate results after deconvolving the data with an
>>> appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist
-
>>> sampled image will be at least 4 (more likely 5) pixels wide, all the
>> noise
>>> terms affect single pixel values. i.e., they have frequency components
at
>>> least 4x higher than that which can represent any real structure. In
>> scanned
>>> fluorescent imaging, one deconvolves more to reduce noise than to
>> increase “
>>> spatial resolution” (although you can also increase resolution as long
as
>>> you have massive amounts of signal.)
>>>
>>> Cheers,
>>>
>>> Jim Pawley
>>> ****************************************
>>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
BC,

>>> Canada, V0N3A0,
>>> Phone [phone], email <[hidden email]<mailto:[hidden email]
>>> <mailto:[hidden email]>>
>>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
>> 1-604-989-6146
>>>
>>>
>>> On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:
>> [hidden email]><mailto:mmodel@
>>> KENT.EDU<http://KENT.EDU>>> 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.
>>> *****
>>>
>>> We have potential users who want to quantify some kind of small
>> aggregates
>>> in the brain. I am afraid that deconvolution can make noise look like
>> such
>>> aggregates. Perhaps collecting a noisy image twice and comparing two
>>> deconvolved images might help, but that seems too much work. Am I wrong?

>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List <[hidden email]<mailto:

>> [hidden email]>> On Behalf
>>> Of Steffen Dietzel
>>> Sent: Friday, May 11, 2018 10:49 AM
>>> To: [hidden email]<mailto:
>> [hidden email]>
>>> Subject: Re: confocal detectors and deconvolution
>>>
>>> *****
>>> 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.
>>> *****
>>>
>>> Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
>>> there is no big difference between HyDs and GaAsP detectors.
>>>
>>> I disagree on this. In my view, the major difference is that the HyD
>> always
>>> operates in photon counting mode whether, as far as I know, the PMTs
>> (with
>>> or without GaAsP) create an electron cloud of which the size is
>> determined
>>> by the number of photoelectrons AND statistics, and the cloud size is
>> then
>>> digitized. So the output created by one photon may vary substantially
>>> depending on the number of electrons created on the first dynodes (which

>> in
>>> turn is a statistical process). My information may be outdated and newer

>>> PMTs might have extra tricks, if so please correct me.
>>>
>>> Another difference is apparently the size of the photcathode. If memory
>>> serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs)

>>> creates more dark noise. I like our HyDs a lot, I appreciate having a
>> gray
>>> value of "21 photons" instead of some random number. But having said
>> this,
>>> at the end of the day what counts is the sensitivity of the whole
system,
>>> and not of the detector alone. So to do this right there is no
substitute
>>> for testing your own samples on different machines with your
>> applications in
>>> mind.
>>>
>>> As for deconvolution, yes, it can create artefacts. But so does confocal

>>> microscopy (a point becomes an Airy pattern, not a point). And if you do

>> it
>>> right the deconvolved image will be closer to the truth than the
original

>>> image. Should you have the third edition of the handbook around, have a
>> look
>>> at the preface, last paragraph.
>>>
>>> Steffen
>>>
>>> --
>>> ------------------------------------------------------------
>>> Steffen Dietzel, PD Dr. rer. nat
>>> Ludwig-Maximilians-Universität München
>>> Biomedical Center (BMC)
>>> Head of the Core Facility Bioimaging
>>>
>>> Großhaderner Straße 9
>>> D-82152 Planegg-Martinsried
>>> Germany
>>>
>>> http://www.bioimaging.bmc.med.uni-muenchen.de
>>>
>>> "
>>>
>>

"

[Craig Brideau]

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Interesting comment Jim! I immediately though of the parallel of scattering
being the bane of confocal as well, so I assume any attempt to descan,
regardless of precision, will be strongly negatively impacted by scattering
as per your analysis. Even a 'loose' descanning as I suggested would be
influenced by this, although not to the degree of a pinhole in a confocal.
Regarding your comment on detector placement, I wonder if a SiPM sensor
could be placed under the sample as it would be much smaller than a PMT...

Craig

On Wed, May 16, 2018 at 10:38 PM JAMES B 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.
> *****
>
> Hi Craig,
>
> Your analysis is correct BUT only for light that proceeds in straight
> lines from the in-focus plane.
>
> Non-rescanned detection is usually used with 2P microscopy to detect light
> that was excited at a particular time (rather than location, as this has
> been defined by the 2P excitation itself). Using time alone to decode
> location allows one to utilize signal photons that have been scattered
> before reaching the objective. This works optimally if you place a PMT that
> is totally insensitive to the 2P excitation directly under the specimen.
> Few systems manage this and the fallback is to collect light that enters
> the objective. You don’t need much optical analysis to show that light
> emerging from a scattering event a few tens of µm towards the objective
> will emerge from this lens as a rapidly diverging ray bundle that doesn’t
> in any way concentrate at the BFP. Any optical components (apart from a
> light pipe) used to try to capture any such rays will only make the
> situation worse.
>
> Even large SiPM  sensors are only 6 mm on a side, considerably smaller
> than the BFP of a 40x NA.1.2NA objective. Consequently, as installing a
> "beam-splitter photodetector" immediately behind the objective is
> difficult, PMTs with photocathodes many mm in diameter work best for this
> sort of methode-non-descanned detection because they can intercept more of
> this rapidly-diverging beam.
>
> Best,
>
> Jim P.
>               ****************************************
> 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
>
> > On May 15, 18, at 11:06 PM, Craig Brideau <[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.
> > *****
> >
> > When designing an optical system for 2P (or confocal for that matter) the
> > scanned beam has to be pivoted around a virtual point located at the back
> > aperture of the objective. If this plane was relayed to the face of the
> > large area detector you would effectively have a descanned detection
> path,
> > loosely speaking.
> >
> > Craig
> >
> > On Tue, May 15, 2018 at 7:25 PM Michael Giacomelli <[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 James,
> >>
> >> That is a very interesting post.  I've read about SiPMs before, but
> >> did not realize they were so far as long. The need to evenly
> >> illuminate a large area is slightly annoying (probably not going to
> >> work well for non-descanned 2P), but could be designed around for a
> >> descanned system as you suggest.  The specs certainly look
> >> interesting, especially the decreased sensitivity to strong light, and
> >> the very low multiplicative noise.
> >>
> >> I wonder if anyone has tried one in a scanning confocal or 2p system
> >> and compared to conventional PMTs (e.g. h7422s or similar)?  I did a
> >> quick search, but didn't see many papers using them for conventional
> >> confocal scanning.
> >>
> >> Mike
> >>
> >> On Mon, May 14, 2018 at 10:50 PM, JAMES B 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.
> >>> *****
> >>>
> >>> Hi Zdenek,
> >>>
> >>> Nice slide show reference! Thanks. I will steal some images!
> >>>
> >>> However…
> >>>
> >>> I think it is a shame that they do not really discuss the difference in
> >> “excess noise” between PMTs and SiPMs as this can affect effective QE
> very
> >> strongly.
> >>>
> >>> Also, I think that they could have done a bit more to separate
> >> applications involving diffuse light from those involving
> almost-coherent
> >> light, like scanning microscopy, in which the signal originates from a
> very
> >> small volume (and therefore can be focussed onto any surface that is
> even
> >> slightly bigger). Of course, PMTs can have huge photocathodes (the
> R877-100
> >> is 12,860 mm2) and, depending on their temp and IR sensitivity, can have
> >> very low dark count rates per square mm of photocathode. But in scanning
> >> light microscopy, there seems to be little need for many square mm of
> >> active area.
> >>>
> >>> And even PMTs can have dark current problems. The first version of the
> >> Zeiss 510 used a large number (23? Don’t remember exactly.) of tiny
> >> servo-motors in which the windings were always excited. This heated the
> >> inside of the optics box enough to produce so many dark counts from
> their
> >> Hamamatsu PMT modules that they had to replace the servos with DC
> versions
> >> that were only excited when you changed some setting. And this was long
> >> before GaAsP photocathodes were common. From slide 19, you can see that
> the
> >> dark count rate of GaAsP is about 100x that of the more common bi-alkali
> >> PMTs.
> >>>
> >>> All the same, silicon diodes do leak current at room temperature and so
> >> it is not surprising that most MPPCs (or SiPMs) are  supplied with
> Peltier
> >> cooling systems.
> >>>
> >>> For most low-light, confocal applications it would seem that the S14420
> >> series might work. It has a 1.5 x 1.5 mm array containing 900, 50µm
> cells
> >> with a fill factor of 81%, a PDE of 40% and a maximum dark count of
> 1,000
> >> cps (or only 0.01 count per (long!) 10µs pixel).
> >>>
> >>>
> https://www.hamamatsu.com/resources/pdf/ssd/s14420_series_kapd1061e.pdf
> >>>
> >>> I know that a max PDE of 40% might sound less than the 40% QE on the
> >> plots of GaAsP PMT photocathodes, but these are different specs: PMT QE
> >> only refers to the fraction of photons making photoelectrons. About 30%
> of
> >> these photoelectrons fail to propagate down the electron-multiplier
> chain
> >> (i.e., they are lost to the signal). If you also include the fact that
> the
> >> MPPC has virtually no excess noise, this makes at least an additional
> 40%
> >> improvement in the effective QE.
> >>>
> >>> BUT you have to make sure that the ray bundle fills the array.
> >>>
> >>> (OK, maybe you don’t. Let’s assume that 600 of the 900 APDs on this
> >> device fall within a circle that just touches the sides of the square
> and
> >> that your signal ray-bundle just fills this circle (uniformly!) when
> your
> >> pinhole is open all the way. If we guess (from Slide 22 of the slide
> show)
> >> that the effective RC decay time is 40ns and choose a pixel time of 1 µs
> >> (or 25 decay times), a simple-minded analysis would lead us to conclude
> >> that a (massive) signal of 600 +/-24 detected photons/1 µs-pixel would
> lose
> >> close to 25 photon pulses due to pulse pile-up (i.e., a number similar
> the
> >> Poisson Noise uncertainty). A detected signal of 64 +/- 8 photons/1µs
> pixel
> >> from a darker area would lose less than one count, an error much smaller
> >> than the Poisson noise.
> >>>
> >>> However, if you keep the same signal levels but use a pinhole that is
> >> 3.3 x smaller in diameter, the signal will now hit only 60 APDs and a
> 600
> >> PE signal will lose 40% because of pulse pile-up. On the other hand, if
> the
> >> signal getting through the smaller pinhole is has gone down with the
> >> pinhole area and is now only 64 +/-8 pulses, then the loss is only 4%,
> or
> >> about 3 counts; smaller than the Poisson noise. For 10µs pixels, the
> signal
> >> would go up by 10 times but the percentage loss would be the same..
> >>>
> >>> So using this detector, you would have another thing to worry about:
> “At
> >> this signal level and this pinhole size, am I losing signal to pileup?”
> >>>
> >>> Annoying, but a geometric factor that the computer could easily keep
> >> track of and warn you when there was a problem.
> >>>
> >>> A worse problem is that, if the bundle diameter is larger than the
> >> back-projected image of the Airy Disk, the light in the bundle is NOT
> >> evenly distributed  As long as there isn’t much spherical aberration,
> the
> >> image of the spot at the pinhole is always a LOT brighter in the
> centre. So
> >> this might be a potential problem, one that could be reduced by using a
> >> larger array (3x3 or 6x6 mm) and changing the optics to fill these
> arrays.
> >> Larger arrays cost more and would increase dark counts by about 4x or
> 16x
> >> (resp.) but probably still not enough to worry about.
> >>>
> >>> Happy designing.
> >>>
> >>> Jim Pawley
> >>>
> >>>              ****************************************
> >>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
> >> BC, Canada, V0N3A0,
> >>> Phone [phone], email <[hidden email]<mailto:[hidden email]
> >>
> >>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> >> 1-604-989-6146
> >>>
> >>> On May 14, 18, at 9:23 AM, [hidden email]<mailto:[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<http://www.imgur.com/> and include
> >> the link in your posting.
> >>> *****
> >>>
> >>>
> >>> To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call
> >> them),
> >>> note that their dark noise is some 3 - 4 orders of magnitude higher
> than
> >>> that of regular PMTs! Of course, chilling the detector to -80 degC
> >> (which is
> >>> common with EMCCDs, for example) would solve this issue, but the cost
> >> would
> >>> be prohibitive...
> >>> For more details on SiPMs vs PMTs see here:
> >>>
> >>> https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB
> >>>
> >>>
> >>> Cheers, zdenek
> >>>
> >>>
> >>>
> >>> ---------- Původní e-mail ----------
> >>> Od: JAMES B PAWLEY <[hidden email]<mailto:[hidden email]>>
> >>> Komu: [hidden email]<mailto:
> >> [hidden email]>
> >>> Datum: 14. 5. 2018 0:27:35
> >>> Předmět: Re: confocal detectors and deconvolution
> >>> "*****
> >>> 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<http://www.imgur.com/> and include
> >> the link in your posting.
> >>> *****
> >>>
> >>> Hi all,
> >>>
> >>> I would like to Eecho Michael’s points.
> >>>
> >>> Because hybrid photodetectors have a very high gain (>>1,000x) in their
> >>> first stage, they produce very little “excess noise” (also called
> >>> Multiplicative Noise). As a result, it is possible to characterize
> their
> >>> output as "25 photons detected” (although it might be safer to think of
> >> it
> >>> as "25 photons detected this time" or ”25 +/- 5 photons.").
> >>>
> >>> Straight PMTs do not share this feature and because single
> photoelectrons
> >>> produce output pulses that vary significantly in size, even the very
> best
> >>> PMTs produce an uncertainty in the magnitude of the signal presented to
> >> the
> >>> ADC that is at least 40% larger in relative terms than would be the
> case
> >> in
> >>> the absence of this excess noise. On PMTs having electron multipliers
> >>> optimized for other reasons (such as making them very small, like those
> >> in
> >>> the 32-PMT linear arrays), the increase in uncertainty is closer to
> 100%
> >> (i.
> >>> e., The signal has the same uncertainty that it would have if 4 times
> >> fewer
> >>> photons were counted perfectly.)
> >>>
> >>> Either type of PMT can have a GaAsP photocathode but it will need to be
> >>> cooled.
> >>>
> >>> Although single APDs may have a high photon detection efficiency (PDE,
> a
> >>> spec that is like QE but which includes the signal lost by
> photoelectrons
> >>> that do not avalanche at all) they have such massive excess noise that
> >> it is
> >>> essential to use them with pulse-counting circuits and these circuits
> are
> >>> just too slow for use in beam-scanning light microscopy.
> >>>
> >>> The solution is the multi-pixel photon counter (MPPC, a development
> from
> >> the
> >>> SPAD (single photon avalanche device),
> >>>
> >>> https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf
> >>>
> >>>
> >>
> https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_
> >>> ultimate_photosensor/index.html ).
> >>>
> >>> The surface of an MPPC is covered with an array of 400 to 20,000 APDs,
> >> each
> >>> connected to the high-voltage rail through its own damping resister.
> The
> >>> resistor causes the voltage across the APD to drop as the avalanche
> >>> proceeds. This quenches the discharge and produces single-photon pulses
> >> of
> >>> very uniform size. As all the APDs are electrically in parallel, these
> >>> single-photon current pulses simply add up producing an output current
> >>> signal almost devoid of excess noise. What could be better? And in
> >> addition
> >>> they are significantly less sensitive than hybrid PMTs to overheating
> >> damage
> >>> if accidentally exposed to a bright light.
> >>>
> >>> There are of course limitations: 1) A significant fraction (20-40%) of
> >> the
> >>> MPPC's surface is taken up with the resistors and the wiring to provide
> >> each
> >>> APD with + and - voltages. Photons absorbed or reflected in these areas
> >> are
> >>> lost. 2) The system is only free of pulse-pileup losses to the extent
> >> that
> >>> no APD absorbs more than one photon within its RC relaxation time (set
> by
> >>> the R of the resistor, and the capacitance (C) of the sensitive area of
> >> the
> >>> APD. Larger individual APDs “waste” proportionally fewer photons
> hitting
> >> the
> >>> resistor and wiring, (increasing their effective QE) but this increases
> >>> their capacitance (making them more susceptible to pulse-pileup).
> >>>
> >>> All will be well as long as the number of photons absorbed in the
> active
> >>> areas during time period RC is small (5%?) compared to the number of
> >> APDs in
> >>> the array THAT ARE ILLUMINATED BY THE BEAM.
> >>>
> >>> The caps above are to remind everyone that, to work properly, the size
> of
> >>> the ray bundle striking the MPPC must be matched to the size of the APD
> >>> array (1.3 to 6 mm square). This can be a problem if we imagine the ray
> >>> bundle being limited by a confocal aperture that can be varied in size
> >> over
> >>> a substantial range. (Do we need a zoom lens to make all possible
> signal
> >> ray
> >>> -bundles match the size of the MPPC array?)
> >>>
> >>> Apart from this, I would like to second the comment that deep imaging
> is
> >>> usually limited by spherical aberration and systems that can correct
> for
> >>> this without “bumping the specimen while you try to adjust the collar”
> >> are
> >>> to be preferred.
> >>>
> >>> I would also like to reaffirm that, assuming the pixel size meets
> >> Nyquist,
> >>> you should ONLY evaluate results after deconvolving the data with an
> >>> appropriate 2D or 3D PSF. Although the smallest real object in a
> Nyquist-
> >>> sampled image will be at least 4 (more likely 5) pixels wide, all the
> >> noise
> >>> terms affect single pixel values. i.e., they have frequency components
> at
> >>> least 4x higher than that which can represent any real structure. In
> >> scanned
> >>> fluorescent imaging, one deconvolves more to reduce noise than to
> >> increase “
> >>> spatial resolution” (although you can also increase resolution as long
> as
> >>> you have massive amounts of signal.)
> >>>
> >>> Cheers,
> >>>
> >>> Jim Pawley
> >>> ****************************************
> >>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
> BC,
> >>> Canada, V0N3A0,
> >>> Phone [phone], email <[hidden email]<mailto:[hidden email]
> >>> <mailto:[hidden email]>>
> >>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> >> 1-604-989-6146
> >>>
> >>>
> >>> On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:
> >> [hidden email]><mailto:mmodel@
> >>> KENT.EDU<http://KENT.EDU>>> 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.
> >>> *****
> >>>
> >>> We have potential users who want to quantify some kind of small
> >> aggregates
> >>> in the brain. I am afraid that deconvolution can make noise look like
> >> such
> >>> aggregates. Perhaps collecting a noisy image twice and comparing two
> >>> deconvolved images might help, but that seems too much work. Am I
> wrong?
> >>>
> >>> -----Original Message-----
> >>> From: Confocal Microscopy List <[hidden email]
> <mailto:
> >> [hidden email]>> On Behalf
> >>> Of Steffen Dietzel
> >>> Sent: Friday, May 11, 2018 10:49 AM
> >>> To: [hidden email]<mailto:
> >> [hidden email]>
> >>> Subject: Re: confocal detectors and deconvolution
> >>>
> >>> *****
> >>> 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.
> >>> *****
> >>>
> >>> Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
> >>> there is no big difference between HyDs and GaAsP detectors.
> >>>
> >>> I disagree on this. In my view, the major difference is that the HyD
> >> always
> >>> operates in photon counting mode whether, as far as I know, the PMTs
> >> (with
> >>> or without GaAsP) create an electron cloud of which the size is
> >> determined
> >>> by the number of photoelectrons AND statistics, and the cloud size is
> >> then
> >>> digitized. So the output created by one photon may vary substantially
> >>> depending on the number of electrons created on the first dynodes
> (which
> >> in
> >>> turn is a statistical process). My information may be outdated and
> newer
> >>> PMTs might have extra tricks, if so please correct me.
> >>>
> >>> Another difference is apparently the size of the photcathode. If memory
> >>> serves me right, the larger cathode of the GaAsP PMTs (compared to
> HyDs)
> >>> creates more dark noise. I like our HyDs a lot, I appreciate having a
> >> gray
> >>> value of "21 photons" instead of some random number. But having said
> >> this,
> >>> at the end of the day what counts is the sensitivity of the whole
> system,
> >>> and not of the detector alone. So to do this right there is no
> substitute
> >>> for testing your own samples on different machines with your
> >> applications in
> >>> mind.
> >>>
> >>> As for deconvolution, yes, it can create artefacts. But so does
> confocal
> >>> microscopy (a point becomes an Airy pattern, not a point). And if you
> do
> >> it
> >>> right the deconvolved image will be closer to the truth than the
> original
> >>> image. Should you have the third edition of the handbook around, have a
> >> look
> >>> at the preface, last paragraph.
> >>>
> >>> Steffen
> >>>
> >>> --
> >>> ------------------------------------------------------------
> >>> Steffen Dietzel, PD Dr. rer. nat
> >>> Ludwig-Maximilians-Universität München
> >>> Biomedical Center (BMC)
> >>> Head of the Core Facility Bioimaging
> >>>
> >>> Großhaderner Straße 9
> >>> D-82152 Planegg-Martinsried
> >>> Germany
> >>>
> >>> http://www.bioimaging.bmc.med.uni-muenchen.de
> >>>
> >>> "
> >>>
> >>
>
>

[Guy Hagen]

*****
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 Colleagues:
Whenever this debate comes up I like to remind that the biggest mistake
people seem to make when trying to use deconvolution in confocal microscopy
is that they have the spatial sampling way too large. In the case of a
confocal microscope with a 1.4 NA objective, the proper sampling is about
50 nm in X,Y and 150 nm in Z. The matter is explained in this excellent
paper by Rainer Heintzmann:

https://www.researchgate.net/publication/47867069_Band_Limit_and_Appropriate_Sampling_in_Microscopy

The reason for this is that the limit (in terms of wavelength) of the
frequency space support for a widefield fluorescence microscope is
Lambda/2NA, whereas for a confocal it is Lambda/4NA. Therefore the sampling
for confocal has to be Lambda/8NA, or about 500/(8*1.4) = 45 nm.

Also i would like to remind that deconvolution is a technique in digital
image processing that is now decades old, and these methods do actually
work. However there are some important "tips and tricks," one being the
re-scaling of intensities in the result. This is usually done because the
deconvolved image can have values that go over the original range (probably
16 bit). If this happens, the image might not be displayed correctly
(especially in programs like photoshop), with over-range values either
being truncated or wrapped around, possibly giving some really strange
looking results.

PS - about photon counting detectors: Traditionally the limit for photon
counting is about 5MHz because of pile-up, in which photon signals start to
bunch together and are no longer counted one at a time. If we are scanning
at 1 image per second, this means that you can only get 5 million photons
per image. A typical confocal microscope image might be much brighter than
this, and so older photon counting methods may not work correctly. Maybe
now there are photon counting setups that can handle much larger count
rates (I guess FLIM is sort of its own story with the 'histogramming'
approach used by B&H).

best,
Guy Hagen




On Thu, May 17, 2018 at 8:22 AM, <[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 James,
> the way I couple detectors to the objective for non-descanned 2P imaging
> is
> by relaying the BFP of the objective lens to the active area of the
> detector. I do it because (1) you couple (almost) all the light that the
> objective lens is able to collect, and (2) the illumination of the
> detector
> is quite homogeneous (the sample itself is perfectly blurred on the
> detector).
>
> With your lens (40x / 1.2 NA) as an example (assuming Oil, Zeiss) with BFP
> diameter around 10 mm, and Hamamatsu hybrid detector with 3 mm diameter
> active area, a simple telecentric relay (f1 = 156 mm, f2 = 50 mm) will do
> the trick. The only question is, how big the two lenses should be? This of
> course depends on the required field of view, and it turns out to be
> (paraxial approcxiamtion, 22 mm field number) 32 mm and 25 mm diameters
> of
> the two relay lenses. Now accounting for the defoucus of several tens of
> µm
> due to light scattering, the relay lenses need to be bigger, but not
> dramatically. For defocus of +/-1% of the objective focal length the
> lenses
> grow to 36 mm and 29 mm diameter. Possibly less with more advanced design.
>
>
> Of course to couple ALL the light that can possibly pass through the
> objective, even bigger relay lenses, or different approach, would be
> needed,
> but that depends on the particularities of each individual objective
> lens...
>
> Best, zdenek
> --
> Zdenek Svindrych, Ph.D.
> Research Associate - Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
> email: [hidden email]
>
> ---------- Původní e-mail ----------
> Od: JAMES B PAWLEY <[hidden email]>
> Komu: [hidden email]
> Datum: 17. 5. 2018 0:42:27
> Předmět: Re: confocal detectors and deconvolution
> "*****
> 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 Craig,
>
> Your analysis is correct BUT only for light that proceeds in straight
> lines
> from the in-focus plane.
>
> Non-rescanned detection is usually used with 2P microscopy to detect light
> that was excited at a particular time (rather than location, as this has
> been defined by the 2P excitation itself). Using time alone to decode
> location allows one to utilize signal photons that have been scattered
> before reaching the objective. This works optimally if you place a PMT
> that
> is totally insensitive to the 2P excitation directly under the specimen.
> Few
> systems manage this and the fallback is to collect light that enters the
> objective. You don’t need much optical analysis to show that light
> emerging
> from a scattering event a few tens of µm towards the objective will emerge
> from this lens as a rapidly diverging ray bundle that doesn’t in any way
> concentrate at the BFP. Any optical components (apart from a light pipe)
> used to try to capture any such rays will only make the situation worse.
>
> Even large SiPM sensors are only 6 mm on a side, considerably smaller than
> the BFP of a 40x NA.1.2NA objective. Consequently, as installing a "beam-
> splitter photodetector" immediately behind the objective is difficult,
> PMTs
> with photocathodes many mm in diameter work best for this sort of methode-
> non-descanned detection because they can intercept more of this rapidly-
> diverging beam.
>
> Best,
>
> Jim P.
> ****************************************
> 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
>
>
> > On May 15, 18, at 11:06 PM, Craig Brideau <[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.
> > *****
> >
> > When designing an optical system for 2P (or confocal for that matter)
> the
> > scanned beam has to be pivoted around a virtual point located at the
> back
> > aperture of the objective. If this plane was relayed to the face of the
> > large area detector you would effectively have a descanned detection
> path,
>
> > loosely speaking.
> >
> > Craig
> >
> > On Tue, May 15, 2018 at 7:25 PM Michael Giacomelli <[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 James,
> >>
> >> That is a very interesting post. I've read about SiPMs before, but
> >> did not realize they were so far as long. The need to evenly
> >> illuminate a large area is slightly annoying (probably not going to
> >> work well for non-descanned 2P), but could be designed around for a
> >> descanned system as you suggest. The specs certainly look
> >> interesting, especially the decreased sensitivity to strong light, and
> >> the very low multiplicative noise.
> >>
> >> I wonder if anyone has tried one in a scanning confocal or 2p system
> >> and compared to conventional PMTs (e.g. h7422s or similar)? I did a
> >> quick search, but didn't see many papers using them for conventional
> >> confocal scanning.
> >>
> >> Mike
> >>
> >> On Mon, May 14, 2018 at 10:50 PM, JAMES B 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.
> >>> *****
> >>>
> >>> Hi Zdenek,
> >>>
> >>> Nice slide show reference! Thanks. I will steal some images!
> >>>
> >>> However…
> >>>
> >>> I think it is a shame that they do not really discuss the difference
> in
> >> “excess noise” between PMTs and SiPMs as this can affect effective QE
> very
> >> strongly.
> >>>
> >>> Also, I think that they could have done a bit more to separate
> >> applications involving diffuse light from those involving
> almost-coherent
>
> >> light, like scanning microscopy, in which the signal originates from a
> very
> >> small volume (and therefore can be focussed onto any surface that is
> even
>
> >> slightly bigger). Of course, PMTs can have huge photocathodes (the R877-
> 100
> >> is 12,860 mm2) and, depending on their temp and IR sensitivity, can
> have
> >> very low dark count rates per square mm of photocathode. But in
> scanning
> >> light microscopy, there seems to be little need for many square mm of
> >> active area.
> >>>
> >>> And even PMTs can have dark current problems. The first version of the
> >> Zeiss 510 used a large number (23? Don’t remember exactly.) of tiny
> >> servo-motors in which the windings were always excited. This heated the
> >> inside of the optics box enough to produce so many dark counts from
> their
>
> >> Hamamatsu PMT modules that they had to replace the servos with DC
> versions
> >> that were only excited when you changed some setting. And this was long
> >> before GaAsP photocathodes were common. From slide 19, you can see that
> the
> >> dark count rate of GaAsP is about 100x that of the more common
> bi-alkali
> >> PMTs.
> >>>
> >>> All the same, silicon diodes do leak current at room temperature and
> so
> >> it is not surprising that most MPPCs (or SiPMs) are supplied with
> Peltier
>
> >> cooling systems.
> >>>
> >>> For most low-light, confocal applications it would seem that the
> S14420
> >> series might work. It has a 1.5 x 1.5 mm array containing 900, 50µm
> cells
>
> >> with a fill factor of 81%, a PDE of 40% and a maximum dark count of
> 1,000
>
> >> cps (or only 0.01 count per (long!) 10µs pixel).
> >>>
> >>> https://www.hamamatsu.com/resources/pdf/ssd/s14420_
> series_kapd1061e.pdf
> >>>
> >>> I know that a max PDE of 40% might sound less than the 40% QE on the
> >> plots of GaAsP PMT photocathodes, but these are different specs: PMT QE
> >> only refers to the fraction of photons making photoelectrons. About 30%
> of
> >> these photoelectrons fail to propagate down the electron-multiplier
> chain
>
> >> (i.e., they are lost to the signal). If you also include the fact that
> the
> >> MPPC has virtually no excess noise, this makes at least an additional
> 40%
>
> >> improvement in the effective QE.
> >>>
> >>> BUT you have to make sure that the ray bundle fills the array.
> >>>
> >>> (OK, maybe you don’t. Let’s assume that 600 of the 900 APDs on this
> >> device fall within a circle that just touches the sides of the square
> and
>
> >> that your signal ray-bundle just fills this circle (uniformly!) when
> your
>
> >> pinhole is open all the way. If we guess (from Slide 22 of the slide
> show)
> >> that the effective RC decay time is 40ns and choose a pixel time of 1
> µs
> >> (or 25 decay times), a simple-minded analysis would lead us to conclude
> >> that a (massive) signal of 600 +/-24 detected photons/1 µs-pixel would
> lose
> >> close to 25 photon pulses due to pulse pile-up (i.e., a number similar
> the
> >> Poisson Noise uncertainty). A detected signal of 64 +/- 8 photons/1µs
> pixel
> >> from a darker area would lose less than one count, an error much
> smaller
> >> than the Poisson noise.
> >>>
> >>> However, if you keep the same signal levels but use a pinhole that is
> >> 3.3 x smaller in diameter, the signal will now hit only 60 APDs and a
> 600
>
> >> PE signal will lose 40% because of pulse pile-up. On the other hand, if
> the
> >> signal getting through the smaller pinhole is has gone down with the
> >> pinhole area and is now only 64 +/-8 pulses, then the loss is only 4%,
> or
>
> >> about 3 counts; smaller than the Poisson noise. For 10µs pixels, the
> signal
> >> would go up by 10 times but the percentage loss would be the same..
> >>>
> >>> So using this detector, you would have another thing to worry about:
> “At
>
> >> this signal level and this pinhole size, am I losing signal to pileup?”
> >>>
> >>> Annoying, but a geometric factor that the computer could easily keep
> >> track of and warn you when there was a problem.
> >>>
> >>> A worse problem is that, if the bundle diameter is larger than the
> >> back-projected image of the Airy Disk, the light in the bundle is NOT
> >> evenly distributed As long as there isn’t much spherical aberration,
> the
> >> image of the spot at the pinhole is always a LOT brighter in the
> centre.
> So
> >> this might be a potential problem, one that could be reduced by using a
> >> larger array (3x3 or 6x6 mm) and changing the optics to fill these
> arrays.
> >> Larger arrays cost more and would increase dark counts by about 4x or
> 16x
>
> >> (resp.) but probably still not enough to worry about.
> >>>
> >>> Happy designing.
> >>>
> >>> Jim Pawley
> >>>
> >>> ****************************************
> >>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
> >> BC, Canada, V0N3A0,
> >>> Phone [phone], email <[hidden email]<mailto:[hidden email]>>
>
> >>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> >> 1-604-989-6146
> >>>
> >>> On May 14, 18, at 9:23 AM, [hidden email]<mailto:[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<http://www.imgur.com/> and
> include
> >> the link in your posting.
> >>> *****
> >>>
> >>>
> >>> To chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call
> >> them),
> >>> note that their dark noise is some 3 - 4 orders of magnitude higher
> than
>
> >>> that of regular PMTs! Of course, chilling the detector to -80 degC
> >> (which is
> >>> common with EMCCDs, for example) would solve this issue, but the cost
> >> would
> >>> be prohibitive...
> >>> For more details on SiPMs vs PMTs see here:
> >>>
> >>> https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB
> >>>
> >>>
> >>> Cheers, zdenek
> >>>
> >>>
> >>>
> >>> ---------- Původní e-mail ----------
> >>> Od: JAMES B PAWLEY <[hidden email]<mailto:[hidden email]>>
> >>> Komu: [hidden email]<mailto:
> >> [hidden email]>
> >>> Datum: 14. 5. 2018 0:27:35
> >>> Předmět: Re: confocal detectors and deconvolution
> >>> "*****
> >>> 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<http://www.imgur.com/> and
> include
> >> the link in your posting.
> >>> *****
> >>>
> >>> Hi all,
> >>>
> >>> I would like to Eecho Michael’s points.
> >>>
> >>> Because hybrid photodetectors have a very high gain (>>1,000x) in
> their
> >>> first stage, they produce very little “excess noise” (also called
> >>> Multiplicative Noise). As a result, it is possible to characterize
> their
>
> >>> output as "25 photons detected” (although it might be safer to think
> of
> >> it
> >>> as "25 photons detected this time" or ”25 +/- 5 photons.").
> >>>
> >>> Straight PMTs do not share this feature and because single
> photoelectrons
> >>> produce output pulses that vary significantly in size, even the very
> best
> >>> PMTs produce an uncertainty in the magnitude of the signal presented
> to
> >> the
> >>> ADC that is at least 40% larger in relative terms than would be the
> case
>
> >> in
> >>> the absence of this excess noise. On PMTs having electron multipliers
> >>> optimized for other reasons (such as making them very small, like
> those
> >> in
> >>> the 32-PMT linear arrays), the increase in uncertainty is closer to
> 100%
>
> >> (i.
> >>> e., The signal has the same uncertainty that it would have if 4 times
> >> fewer
> >>> photons were counted perfectly.)
> >>>
> >>> Either type of PMT can have a GaAsP photocathode but it will need to
> be
> >>> cooled.
> >>>
> >>> Although single APDs may have a high photon detection efficiency (PDE,
> a
>
> >>> spec that is like QE but which includes the signal lost by
> photoelectrons
> >>> that do not avalanche at all) they have such massive excess noise that
> >> it is
> >>> essential to use them with pulse-counting circuits and these circuits
> are
> >>> just too slow for use in beam-scanning light microscopy.
> >>>
> >>> The solution is the multi-pixel photon counter (MPPC, a development
> from
>
> >> the
> >>> SPAD (single photon avalanche device),
> >>>
> >>> https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf
> >>>
> >>>
> >> https://www.hamamatsu.com/us/en/community/optical_sensors/
> articles/sipm_
> the_
> >>> ultimate_photosensor/index.html ).
> >>>
> >>> The surface of an MPPC is covered with an array of 400 to 20,000 APDs,
> >> each
> >>> connected to the high-voltage rail through its own damping resister.
> The
>
> >>> resistor causes the voltage across the APD to drop as the avalanche
> >>> proceeds. This quenches the discharge and produces single-photon
> pulses
> >> of
> >>> very uniform size. As all the APDs are electrically in parallel, these
> >>> single-photon current pulses simply add up producing an output current
> >>> signal almost devoid of excess noise. What could be better? And in
> >> addition
> >>> they are significantly less sensitive than hybrid PMTs to overheating
> >> damage
> >>> if accidentally exposed to a bright light.
> >>>
> >>> There are of course limitations: 1) A significant fraction (20-40%) of
> >> the
> >>> MPPC's surface is taken up with the resistors and the wiring to
> provide
> >> each
> >>> APD with + and - voltages. Photons absorbed or reflected in these
> areas
> >> are
> >>> lost. 2) The system is only free of pulse-pileup losses to the extent
> >> that
> >>> no APD absorbs more than one photon within its RC relaxation time (set
> by
> >>> the R of the resistor, and the capacitance (C) of the sensitive area
> of
> >> the
> >>> APD. Larger individual APDs “waste” proportionally fewer photons
> hitting
>
> >> the
> >>> resistor and wiring, (increasing their effective QE) but this
> increases
> >>> their capacitance (making them more susceptible to pulse-pileup).
> >>>
> >>> All will be well as long as the number of photons absorbed in the
> active
>
> >>> areas during time period RC is small (5%?) compared to the number of
> >> APDs in
> >>> the array THAT ARE ILLUMINATED BY THE BEAM.
> >>>
> >>> The caps above are to remind everyone that, to work properly, the size
> of
> >>> the ray bundle striking the MPPC must be matched to the size of the
> APD
> >>> array (1.3 to 6 mm square). This can be a problem if we imagine the
> ray
> >>> bundle being limited by a confocal aperture that can be varied in size
> >> over
> >>> a substantial range. (Do we need a zoom lens to make all possible
> signal
>
> >> ray
> >>> -bundles match the size of the MPPC array?)
> >>>
> >>> Apart from this, I would like to second the comment that deep imaging
> is
>
> >>> usually limited by spherical aberration and systems that can correct
> for
>
> >>> this without “bumping the specimen while you try to adjust the collar”
> >> are
> >>> to be preferred.
> >>>
> >>> I would also like to reaffirm that, assuming the pixel size meets
> >> Nyquist,
> >>> you should ONLY evaluate results after deconvolving the data with an
> >>> appropriate 2D or 3D PSF. Although the smallest real object in a
> Nyquist
> -
> >>> sampled image will be at least 4 (more likely 5) pixels wide, all the
> >> noise
> >>> terms affect single pixel values. i.e., they have frequency components
> at
> >>> least 4x higher than that which can represent any real structure. In
> >> scanned
> >>> fluorescent imaging, one deconvolves more to reduce noise than to
> >> increase “
> >>> spatial resolution” (although you can also increase resolution as long
> as
> >>> you have massive amounts of signal.)
> >>>
> >>> Cheers,
> >>>
> >>> Jim Pawley
> >>> ****************************************
> >>> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
> BC,
> >>> Canada, V0N3A0,
> >>> Phone [phone], email <[hidden email]<mailto:[hidden email]
> >>> <mailto:[hidden email]>>
> >>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> >> 1-604-989-6146
> >>>
> >>>
> >>> On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:
> >> [hidden email]><mailto:mmodel@
> >>> KENT.EDU<http://KENT.EDU>>> 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.
> >>> *****
> >>>
> >>> We have potential users who want to quantify some kind of small
> >> aggregates
> >>> in the brain. I am afraid that deconvolution can make noise look like
> >> such
> >>> aggregates. Perhaps collecting a noisy image twice and comparing two
> >>> deconvolved images might help, but that seems too much work. Am I
> wrong?
>
> >>>
> >>> -----Original Message-----
> >>> From: Confocal Microscopy List <[hidden email]
> <mailto:
>
> >> [hidden email]>> On Behalf
> >>> Of Steffen Dietzel
> >>> Sent: Friday, May 11, 2018 10:49 AM
> >>> To: [hidden email]<mailto:
> >> [hidden email]>
> >>> Subject: Re: confocal detectors and deconvolution
> >>>
> >>> *****
> >>> 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.
> >>> *****
> >>>
> >>> Am 10.05.2018 um 17:19 schrieb Vitaly Boyko:
> >>> there is no big difference between HyDs and GaAsP detectors.
> >>>
> >>> I disagree on this. In my view, the major difference is that the HyD
> >> always
> >>> operates in photon counting mode whether, as far as I know, the PMTs
> >> (with
> >>> or without GaAsP) create an electron cloud of which the size is
> >> determined
> >>> by the number of photoelectrons AND statistics, and the cloud size is
> >> then
> >>> digitized. So the output created by one photon may vary substantially
> >>> depending on the number of electrons created on the first dynodes
> (which
>
> >> in
> >>> turn is a statistical process). My information may be outdated and
> newer
>
> >>> PMTs might have extra tricks, if so please correct me.
> >>>
> >>> Another difference is apparently the size of the photcathode. If
> memory
> >>> serves me right, the larger cathode of the GaAsP PMTs (compared to
> HyDs)
>
> >>> creates more dark noise. I like our HyDs a lot, I appreciate having a
> >> gray
> >>> value of "21 photons" instead of some random number. But having said
> >> this,
> >>> at the end of the day what counts is the sensitivity of the whole
> system,
> >>> and not of the detector alone. So to do this right there is no
> substitute
> >>> for testing your own samples on different machines with your
> >> applications in
> >>> mind.
> >>>
> >>> As for deconvolution, yes, it can create artefacts. But so does
> confocal
>
> >>> microscopy (a point becomes an Airy pattern, not a point). And if you
> do
>
> >> it
> >>> right the deconvolved image will be closer to the truth than the
> original
> >>> image. Should you have the third edition of the handbook around, have
> a
> >> look
> >>> at the preface, last paragraph.
> >>>
> >>> Steffen
> >>>
> >>> --
> >>> ------------------------------------------------------------
> >>> Steffen Dietzel, PD Dr. rer. nat
> >>> Ludwig-Maximilians-Universität München
> >>> Biomedical Center (BMC)
> >>> Head of the Core Facility Bioimaging
> >>>
> >>> Großhaderner Straße 9
> >>> D-82152 Planegg-Martinsried
> >>> Germany
> >>>
> >>> http://www.bioimaging.bmc.med.uni-muenchen.de
> >>>
> >>> "
> >>>
> >>
>
> "
>

[Giuseppe Vicidomini]

*****
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 friends,
        regarding the topic of confocal detectors and deconvolution I hope that you will find interesting our new preprint work (http://biorxiv.org/cgi/content/short/335596v1 <http://biorxiv.org/cgi/content/short/335596v1>)
This work introduces the use of an asynchronous (no-frame rate) SPAD array in single-point scanning microscopy. SPAD array are not new in the field of fluorescence microscopy, they were used both in wide-field and multi-point scanning microscopy. But, as far as I know, its application in the context of single-point scanning microscopy has been mainly precluded by the limiting characteristics of the current SPAD array  (e.g. as fill-factor, PDE, size, etc). In this work, we demonstrate that these limitations can be overcome and our detector would like to trigger the development of a new generation of SPAD array designed ad-hoc for single-point scanning microscopy (linear and non-linear). I personally believe that one of the key advantage of point-scanning microscopy is its multi-parameter nature, i.e., its possibility to straightforward combine confocal or multi-photon microscopy with spectroscopy (time-, spectral- and polarisation-resolved) and intensity fluctuation analysis (such as FCS). For this reason, maintaining these abilities was an important asset for us.  Last, but not least, we used the extra spatial information provides by the SPAD array to naturally implement image scanning microscopy, whose benefit in term of spatial resolution has been deeply demonstrated in this last years. We also demonstrate the synergistic combination of deconvolution with SPAD array.
Happy to know your opinion.
Best regards
Giuseppe

> On 10 May 2018, at 16:00, MODEL, MICHAEL <[hidden email] <mailto:[hidden email]>> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy <http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear colleagues,
>
>
>
> we are looking for a new confocal microscope, and I have two questions so far.
>
> 1.      Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then.
>
> 2.      Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't  had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra.
>
>
>
> Thank you
>
>
>
> Mike Model
>
> Kent State University

[Zdenek Svindrych-2]

*****
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 Guiseppe,
very interesting work, indeed!

I was surprised to see you call this method "ISM" (after the 2010 Muller and
Enderlein paper), and not Colin Sheppard's favorite "OPRA" :-).

I like the way you use cross-correlation to determine the shift vectors for
individual SPAD pixels. I used the same trick to figure out the arrangement
of the individual channels on the Zeiss' Airyscan honeycomb detector. But
then I used the geometrical vectors, as they seemed to me more precise and
not dependent on the sample frequency content.

But what I found most interesting is the discussion of image processing
methods. The Zeiss technical note ( https://p.widencdn.net/qpx75n ) mentions
the main drawback of the "Sheppard sum" approach, that is, it does not
increase the axial resolution. Enderlein (2010) combined the Sheppard sum
with a simple deconvolution (non iterative Fourier filtering in 2D), still
the axial resolution is just "confocal".


Now Zeiss uses different strategy for Airyscan processing: they first
deconvolve the raw images and then apply the Sheppard sum. The deconvolution
is again simple non-iterative Weiner filter, but the axial resolution is
increased if the filtering is done in 3D!!

A multi-image deconvolution (MLE estimate of the true image based on all the
raw images, with appropriate photon statistics), just like in your preprint,
should be the best method, but to unleash it's full potential, a 3D approach
is mandatory.

I know your paper is focused on the SPAD array, but it would be great to
write a paper comparing these (and other) methods of ISM image processing.
Btw, could anyone shed some light on the "Zeiss new 2S superresolution mode
for Airyscan"?  See https://www.nature.com/articles/nmeth.f.404  -don't get
confused, this is not a Nature Methods paper, just a commercial! 

No commercial interest.

Best, zdenek
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
email: [hidden email]

---------- Původní e-mail ----------
Od: Giuseppe Vicidomini <[hidden email]>
Komu: [hidden email]
Datum: 6. 6. 2018 4:22:15
Předmět: Re: confocal detectors and deconvolution
"*****
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 friends,
regarding the topic of confocal detectors and deconvolution I hope that you
will find interesting our new preprint work (http://biorxiv.org/cgi/content/
short/335596v1 <http://biorxiv.org/cgi/content/short/335596v1>)
This work introduces the use of an asynchronous (no-frame rate) SPAD array
in single-point scanning microscopy. SPAD array are not new in the field of
fluorescence microscopy, they were used both in wide-field and multi-point
scanning microscopy. But, as far as I know, its application in the context
of single-point scanning microscopy has been mainly precluded by the
limiting characteristics of the current SPAD array (e.g. as fill-factor,
PDE, size, etc). In this work, we demonstrate that these limitations can be
overcome and our detector would like to trigger the development of a new
generation of SPAD array designed ad-hoc for single-point scanning
microscopy (linear and non-linear). I personally believe that one of the key
advantage of point-scanning microscopy is its multi-parameter nature, i.e.,
its possibility to straightforward combine confocal or multi-photon
microscopy with spectroscopy (time-, spectral- and polarisation-resolved)
and intensity fluctuation analysis (such as FCS). For this reason,
maintaining these abilities was an important asset for us. Last, but not
least, we used the extra spatial information provides by the SPAD array to
naturally implement image scanning microscopy, whose benefit in term of
spatial resolution has been deeply demonstrated in this last years. We also
demonstrate the synergistic combination of deconvolution with SPAD array.
Happy to know your opinion.
Best regards
Giuseppe


> On 10 May 2018, at 16:00, MODEL, MICHAEL <[hidden email] <mailto:mmodel@
KENT.EDU>> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy <http://lists.umn.
edu/cgi-bin/wa?A0=confocalmicroscopy>
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear colleagues,
>
>
>
> we are looking for a new confocal microscope, and I have two questions so
far.
>
> 1. Are Leica'a hybrid detectors (I am talking about SP8) in any way better
than the standard GaAsP? I found an old discussion of this topic on this
list, but technology may have improved since then.
>
> 2. Both Leica and Olympus 3000 use deconvolution to boost resolution. We
haven't had enough time during the demos to test it thoroughly, but my
impression is that some features "revealed" by deconvolution might be
artefactual, so I don't know if it is worth paying extra.

>
>
>
> Thank you
>
>
>
> Mike Model
>
> Kent State University

"