Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

[Stanislav Vitha-2]

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I am curious what pulsed light sources are being used for the simulated
global shutter mode with sCMOS cameras (that have the 4-transistor
architecture, like Zyla 4.2, Flash4.2, ..).
I am aware of the OptoLED module from Cairn Research, but I remember
seeing a statement on their website that they cannot sell them in the USA
for microscope illumination.

Thanks in advance!

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

[Kurt Thorn]

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We use a Lumencor Spectra-X
(http://lumencor.com/product/spectra-x-light-engine/) driven by an ESIO
controller
(http://www.esimagingsolutions.com/products/esio-controllers), running
Micro-manager.  This works very well using our Zyla 5.5 in rolling
shutter mode.

Kurt

On 5/7/2014 3:07 PM, Stanislav Vitha 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.
> *****
>
> I am curious what pulsed light sources are being used for the simulated
> global shutter mode with sCMOS cameras (that have the 4-transistor
> architecture, like Zyla 4.2, Flash4.2, ..).
> I am aware of the OptoLED module from Cairn Research, but I remember
> seeing a statement on their website that they cannot sell them in the USA
> for microscope illumination.
>
> Thanks in advance!
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University
>
>
>
>
>  
>
>

--
Kurt Thorn
Director, Nikon Imaging Center
http://nic.ucsf.edu/blog/

[Mark A. Sanders]

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We happily use the Zyla 5.5, 10-tap, in rolling shutter mode with TTL commands from the Lumencor Spectra-X (http://lumencor.com/product/spectra-x-light-engine/) with 395, 440, 477, 555, 640 wavelengths all driven by Nikon Elements.

Cheers,
Mark
************************************************
Mark A. Sanders      University of Minnesota
Program Director      Twin Cities Campus
University Imaging Centers  
www.uic.umn.edu
 

On May 7, 2014, at 5:07 PM, Stanislav Vitha <[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.
> *****
>
> I am curious what pulsed light sources are being used for the simulated
> global shutter mode with sCMOS cameras (that have the 4-transistor
> architecture, like Zyla 4.2, Flash4.2, ..).
> I am aware of the OptoLED module from Cairn Research, but I remember
> seeing a statement on their website that they cannot sell them in the USA
> for microscope illumination.
>
> Thanks in advance!
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University
>
>
>
>
>

[Gerhard Holst]

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I guess the pulsed light source just depends on the optics and the light, that the sample requires, whenever you can trigger the light with the camera. In the simulated global shutter mode, like you call it, it is just a speeded up rolling reset, whenever the CIS2020 sCMOS image sensor is used (like in the mentioned Zyla 4.2, Flash 4.0, pco.edge 4.2).
If you have time enough just wait until the reset line has passed the whole sensor, the exposure time is anyhow dominated by the flash.

If you have to follow the pulsed lamp, or if you want to influence the exposure during flash (assuming that that flash or pulse is longer than the shortest exposure time) then the CIS2521 sCMOS cameras (like Zyla 5.5, pco.edge 5.5) can do better, since have due to 5th transistor a real global shutter and as well a global reset and rolling readout.

Nevertheless, I don't see the conditional relation between the fast rolling reset and the pulsed light source, or did I oversee something?

with best regards,

Gerhard Holst
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Stanislav Vitha
Gesendet: Donnerstag, 8. Mai 2014 00:07
An: [hidden email]
Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

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*****

I am curious what pulsed light sources are being used for the simulated
global shutter mode with sCMOS cameras (that have the 4-transistor
architecture, like Zyla 4.2, Flash4.2, ..).
I am aware of the OptoLED module from Cairn Research, but I remember
seeing a statement on their website that they cannot sell them in the USA
for microscope illumination.

Thanks in advance!

Stan Vitha
Microscopy and Imaging Center
Texas A&M University




 

[Doube, Michael]

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On 08/05/14 20:08, Stanislav Vitha wrote:
> I am curious what pulsed light sources are being used for the simulated
> global shutter mode with sCMOS cameras (that have the 4-transistor
> architecture, like Zyla 4.2, Flash4.2, ..).

I have built a fairly agricultural-looking LED trigger box out of very basic components which works for both Zyla and Flash with trigger-outs. Components cost a few pounds altogether, illumination is fast switching on the cameras' trigger-out signal and 'bright enough'. Schematic available off-list if anyone is interested.

Michael

<http://www.rvc.ac.uk>

This message, together with any attachments, is intended for the stated addressee(s) only and may contain privileged or confidential information. Any views or opinions presented are solely those of the author and do not necessarily represent those of the Royal Veterinary College.

[Shalin Mehta]

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Stan,
FWIW, I measured Cairn OptoLED's switching time to be 10us
(http://blog.mshalin.com/2014/03/evaluation-of-optoled-for-emulating_3326.html).
It worked very well with our Flash.  It is interesting to know that
X-Cite XLED1 also provides 10us switching time.

Best
Shalin
Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.


On Wed, May 7, 2014 at 6:07 PM, Stanislav Vitha <[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.
> *****
>
> I am curious what pulsed light sources are being used for the simulated
> global shutter mode with sCMOS cameras (that have the 4-transistor
> architecture, like Zyla 4.2, Flash4.2, ..).
> I am aware of the OptoLED module from Cairn Research, but I remember
> seeing a statement on their website that they cannot sell them in the USA
> for microscope illumination.
>
> Thanks in advance!
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University
>
>
>
>
>

[Gerhard Holst]

Since the "pseudo-Global reset" in the CIS2020 based cameras like Flash 4.0, Zyla 4.2 or pco.edge 4.2 takes at high pixelclock 165.6 us and at low pixelclcock 292.3 us, I am curious, why a short switching time of 10 us of the light source is important for your applications? Just to understand...

with best regards,

Gerhard Holst
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Shalin Mehta
Gesendet: Montag, 12. Mai 2014 22:49
An: [hidden email]
Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

*****
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*****

Stan,
FWIW, I measured Cairn OptoLED's switching time to be 10us
(http://blog.mshalin.com/2014/03/evaluation-of-optoled-for-emulating_3326.html).
It worked very well with our Flash.  It is interesting to know that
X-Cite XLED1 also provides 10us switching time.

Best
Shalin
Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.


On Wed, May 7, 2014 at 6:07 PM, Stanislav Vitha <[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.
> *****
>
> I am curious what pulsed light sources are being used for the simulated
> global shutter mode with sCMOS cameras (that have the 4-transistor
> architecture, like Zyla 4.2, Flash4.2, ..).
> I am aware of the OptoLED module from Cairn Research, but I remember
> seeing a statement on their website that they cannot sell them in the USA
> for microscope illumination.
>
> Thanks in advance!
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University
>
>
>
>
>

[Shalin Mehta]

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

Gerhard, could you elaborate what 'pseudo-global reset' is for CIS2020?

I acquired the data noted here
(http://blog.mshalin.com/2014/03/evaluation-of-optoled-for-emulating_3326.html)
with 1ms effective exposure by setting the camera exposure to 11ms.
10ms taken for all rows to become active and all rows stay active for
1ms.

I would use 250us exposure, had there been enough light (this is
transmitted light microscopy not fluorescence).

If the LED takes 100us to switch from OFF to ON, it will illuminate
the specimen sub-optimally after all rows are active. If it takes
100us to switch from ON to OFF, it will illuminate rows unevenly as
the next exposure starts and introduce slight  rolling-shutter
artifacts.

Switching over 10us (time it takes to activate one row) is useful to
avoid both of the above issues.


Shalin

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.


On Tue, May 13, 2014 at 5:29 AM, Gerhard Holst <[hidden email]> wrote:

> Since the "pseudo-Global reset" in the CIS2020 based cameras like Flash 4.0, Zyla 4.2 or pco.edge 4.2 takes at high pixelclock 165.6 us and at low pixelclcock 292.3 us, I am curious, why a short switching time of 10 us of the light source is important for your applications? Just to understand...
>
> with best regards,
>
> Gerhard Holst
> _______________________________
>
> Dr. Gerhard Holst
> Science & Research
> PCO AG
> Donaupark 11
> 93309 Kelheim, Germany
> fon [phone]
> fax [phone]
> mob [phone]
> [hidden email]
> www.pco.de
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Shalin Mehta
> Gesendet: Montag, 12. Mai 2014 22:49
> An: [hidden email]
> Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?
>
> *****
> 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.
> *****
>
> Stan,
> FWIW, I measured Cairn OptoLED's switching time to be 10us
> (http://blog.mshalin.com/2014/03/evaluation-of-optoled-for-emulating_3326.html).
> It worked very well with our Flash.  It is interesting to know that
> X-Cite XLED1 also provides 10us switching time.
>
> Best
> Shalin
> Assistant Research Scientist,
> Marine Biological Laboratory,
> 7 MBL Street, Woods Hole MA 02543, USA
>
> website: http://mshalin.com
> (office) Lillie 110, (ph) 508-289-7374.
>
>
> On Wed, May 7, 2014 at 6:07 PM, Stanislav Vitha <[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.
>> *****
>>
>> I am curious what pulsed light sources are being used for the simulated
>> global shutter mode with sCMOS cameras (that have the 4-transistor
>> architecture, like Zyla 4.2, Flash4.2, ..).
>> I am aware of the OptoLED module from Cairn Research, but I remember
>> seeing a statement on their website that they cannot sell them in the USA
>> for microscope illumination.
>>
>> Thanks in advance!
>>
>> Stan Vitha
>> Microscopy and Imaging Center
>> Texas A&M University
>>
>>
>>
>>
>>

[Gerhard Holst]

Hi Shalin,

there are the two different sCMOS image sensors the CIS2521 (2560 x 2160 pixel) and the CIS2020 (2048 x 2048 pixel).
The CIS2521 due to his architecture offers 3 different operational modes of the shutter:
1. Rolling shutter (with all possibilities for the 2 sensor halves: top to center readout, center to top readout, one directional readout = half the speed)
2. Global reset, rolling readout (still high speed and low readout noise like in rolling shutter, but increasing dark current from the first to the last row, which is in most cases due to the short exposure time negligible)
3. Global shutter (because the CDS has to be done externally, a dark image and the exposed image have to be read out, charges are stored to be read out in a capacitor, which adds to the noise, resulting in a slightly higher readout noise, and half of the possible speed (because of the 2 images))

The CIS2020 due to his architecture offers 1 operational mode of the shutter:
1. Rolling shutter (with all possibilities for the 2 sensor halves: top to center readout, center to top readout, one directional readout = half the speed)
Because of the interest of some customers in triggering the camera with the light source, the manufacturers of the CIS2020 sCMOS cameras integrated a fast rolling reset and a consecutive slower rolling readout. The issue is that for both sCMOS sensors there are digital electronics on the sensor controlling the timing, this can be changed by loading some registers with new data, but then the sensor does some restart process which costs time. Therefore this pseudo-global reset is as I wrote about 165.6 us (high pixelclock) and 292.3 us (low pixelclock), which is longer than a global reset, but for some applications it helps.

If you can use the trigger signals from the camera it should be possible to trigger the lamp, whenever the last row is reset in rolling shutter operation.

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Shalin Mehta
Gesendet: Mittwoch, 14. Mai 2014 03:20
An: [hidden email]
Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

*****
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*****

Gerhard, could you elaborate what 'pseudo-global reset' is for CIS2020?

I acquired the data noted here
(http://blog.mshalin.com/2014/03/evaluation-of-optoled-for-emulating_3326.html)
with 1ms effective exposure by setting the camera exposure to 11ms.
10ms taken for all rows to become active and all rows stay active for
1ms.

I would use 250us exposure, had there been enough light (this is
transmitted light microscopy not fluorescence).

If the LED takes 100us to switch from OFF to ON, it will illuminate
the specimen sub-optimally after all rows are active. If it takes
100us to switch from ON to OFF, it will illuminate rows unevenly as
the next exposure starts and introduce slight  rolling-shutter
artifacts.

Switching over 10us (time it takes to activate one row) is useful to
avoid both of the above issues.


Shalin

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.


On Tue, May 13, 2014 at 5:29 AM, Gerhard Holst <[hidden email]> wrote:

> Since the "pseudo-Global reset" in the CIS2020 based cameras like Flash 4.0, Zyla 4.2 or pco.edge 4.2 takes at high pixelclock 165.6 us and at low pixelclcock 292.3 us, I am curious, why a short switching time of 10 us of the light source is important for your applications? Just to understand...
>
> with best regards,
>
> Gerhard Holst
> _______________________________
>
> Dr. Gerhard Holst
> Science & Research
> PCO AG
> Donaupark 11
> 93309 Kelheim, Germany
> fon [phone]
> fax [phone]
> mob [phone]
> [hidden email]
> www.pco.de
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Shalin Mehta
> Gesendet: Montag, 12. Mai 2014 22:49
> An: [hidden email]
> Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?
>
> *****
> 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.
> *****
>
> Stan,
> FWIW, I measured Cairn OptoLED's switching time to be 10us
> (http://blog.mshalin.com/2014/03/evaluation-of-optoled-for-emulating_3326.html).
> It worked very well with our Flash.  It is interesting to know that
> X-Cite XLED1 also provides 10us switching time.
>
> Best
> Shalin
> Assistant Research Scientist,
> Marine Biological Laboratory,
> 7 MBL Street, Woods Hole MA 02543, USA
>
> website: http://mshalin.com
> (office) Lillie 110, (ph) 508-289-7374.
>
>
> On Wed, May 7, 2014 at 6:07 PM, Stanislav Vitha <[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.
>> *****
>>
>> I am curious what pulsed light sources are being used for the simulated
>> global shutter mode with sCMOS cameras (that have the 4-transistor
>> architecture, like Zyla 4.2, Flash4.2, ..).
>> I am aware of the OptoLED module from Cairn Research, but I remember
>> seeing a statement on their website that they cannot sell them in the USA
>> for microscope illumination.
>>
>> Thanks in advance!
>>
>> Stan Vitha
>> Microscopy and Imaging Center
>> Texas A&M University
>>
>>
>>
>>
>>

[Sergey Tauger]

*****
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*****

Is it possible to turn off Zyla's onboar FPGA and compare data obtained from the
same sample with and without onboard processing?
 
Sergey Tauger
MSU

[Gerhard Holst]

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Hi Sergey,

I don't want to hairsplit, and I can't answer this about the Zyla, because it is not our camera, but the FPGA does usually all the control, timing, readout of the images and the pre-processing. This pre-processing involves some treatment of the raw data, that should be there to make useful images:
1. Fixed Pattern correction in the dark (=> offset correction, since every column, pixel is a little bit different in offset)
2. Fixed Pattern correction in the bright (=> gain correction, since every column, pixel is a little bit different in gain)
3. Linearization (the raw relationship between incoming photons and outcoming signal is not linear)
4. Hot (dead) Pixel correction, (Usually a list with the position is stored and the pixel value is replaced by a more or less weighted average of the neighbor pixel, this is done in every digital camera), this is something this can be switched off, if required, in many scientific cameras by a certain command, as well in many cameras, this can be read out (keep in mind, there doesn't exist an image sensor without such dead or hot pixels, they are called "hot" because many of them appear with the maximum value in the image, => white = hot pixels)
5. in the sCMOS cameras the manufacturers apply as well a dynamic algorithm to do pretty much the same for the "blinking" pixels, since they appear like "hot" or "warm" pixel, but not in every image, this filter usually can be switched on and off by command.

Now, it is up to your application, what you want to switch off.

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Sergey Tauger
Gesendet: Mittwoch, 14. Mai 2014 17:40
An: [hidden email]
Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

*****
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*****

Is it possible to turn off Zyla's onboar FPGA and compare data obtained from the
same sample with and without onboard processing?
 
Sergey Tauger
MSU

[Zdenek Svindrych]

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Hi Gerhard,



the linearization step surprised me a bit. When we do e.g. a STORM image
analysis, we work with Poisson statistics. This only holds when the camera's
physical response is linear. Can you tell how severe the nonlinear effects
are? At what intensities? I'm particularly interested in CIS2051 based
cameras...




Thanks,

zdenek svindrych

1st faculty of medicine, Charles University






---------- Původní zpráva ----------
Od: Gerhard Holst <[hidden email]>
Komu: [hidden email]
Datum: 15. 5. 2014 8:38:56
Předmět: AW: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 /
Flash4 V2)?

"*****
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 Sergey,

I don't want to hairsplit, and I can't answer this about the Zyla, because
it is not our camera, but the FPGA does usually all the control, timing,
readout of the images and the pre-processing. This pre-processing involves
some treatment of the raw data, that should be there to make useful images:
1. Fixed Pattern correction in the dark (=> offset correction, since every
column, pixel is a little bit different in offset)
2. Fixed Pattern correction in the bright (=> gain correction, since every
column, pixel is a little bit different in gain)
3. Linearization (the raw relationship between incoming photons and
outcoming signal is not linear)
4. Hot (dead) Pixel correction, (Usually a list with the position is stored
and the pixel value is replaced by a more or less weighted average of the
neighbor pixel, this is done in every digital camera), this is something
this can be switched off, if required, in many scientific cameras by a
certain command, as well in many cameras, this can be read out (keep in
mind, there doesn't exist an image sensor without such dead or hot pixels,
they are called "hot" because many of them appear with the maximum value in
the image, => white = hot pixels)
5. in the sCMOS cameras the manufacturers apply as well a dynamic algorithm
to do pretty much the same for the "blinking" pixels, since they appear like
"hot" or "warm" pixel, but not in every image, this filter usually can be
switched on and off by command.

Now, it is up to your application, what you want to switch off.

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im
Auftrag von Sergey Tauger
Gesendet: Mittwoch, 14. Mai 2014 17:40
An: [hidden email]
Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 /
Flash4 V2)?

*****
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*****

Is it possible to turn off Zyla's onboar FPGA and compare data obtained from
the
same sample with and without onboard processing?

Sergey Tauger
MSU"

[Sergey Tauger]

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Gerhard,

Thanks a lot for your reply. The main thing why I wanted to discard FPGA is that I
thought onboard processing makes images less linear. Now I think that my mistake
was choosing a wrong amplifier gain - I got really poor contrast.
http://www.andor.com/learning-academy/dual-amplifier-dynamic-range-scmos-
dynamic-range

Thanks,
Sergey

[Colin Coates]

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**Commercial response**

Hi,

Just to chip in on the question of manipulating the FPGA processing
functionality of the Zyla.

Gerhard indeed gave a good synopsis of what real time processing goes on
in sCMOS cameras, in order to turn raw images into acceptable images. In
theory its possible to crack in and switch stuff like this off, but in most
cases its not recommended. For most part, they are implemented in order
to bring us to the situation we are used to with CCDs, except with lower
noise, faster speed and wider dynamic range of course! That is to say, the
processing is in place in order to provide us with a nice stable and uniform
bias offset, with low read noise sitting on top, and uniform responsivity to
light across the array.

The most fundamental normalisation we do is to patch up the fixed pattern
noise in the bias (baseline) offset. A comparison with and without is shown
on P20 of the Andor sCMOS brochure:



http://www.andor.com/pdfs/literature/Andor_sCMOS_Brochure.pdf



Then there's a further round of real time bias correction called Dynamic
Baseline Clamp.

Gerhard also mentioned gain compensation. This is indeed implemented to
normalise for slight responsivity differences between sCMOS pixels. A
further side effect of not implementing this compensation would be to
discernibly identify the two halves of the sensor when the sensor is
illuminated.



In our experience, the one area of real time processing that can represent
a problem to people's usage, especially in pointillist super-resolution, is
interpolative pixel filtering (referred to by Gerhard in context of hot pixels).
In fact, Andor have now provided a means to switch off all interpolative
filtering, such that no pixel values are replaced. Furthermore, if useful,
we'll work with you to provide maps of where problematic pixels are
located, such that at least you are aware of them. We are close to releasing
a technical note on this to provide further detail and instruction. If you are
interested, please contact my colleague Orla Hanrahan
([hidden email]) for specific detail.



Best,

Colin



Dr Colin Coates

Product Manager

Andor Technology

[George McNamara]

*****
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*****

Hi Colin,

Could you (and Gerhard/PCO and someone from Hamamatsu) please explain
why you are not correcting the gain for every pixel, re: the mess shown
in Huang ... Bewersdorf et al 2013,
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696415/ , supplemental
figure 1,

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696415/bin/NIHMS475969-supplement-1.pdf

This should be doable at the camera device driver level (would be nice
if implemented 'realtime' on a GPU or Intel Phi card) so that everyone
who owns any of your cameras could get this for just the effort of a
driver update.

thanks in advance,

George

On 5/15/2014 11:46 AM, Colin Coates 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.
> *****
>
> **Commercial response**
>
> Hi,
>
> Just to chip in on the question of manipulating the FPGA processing
> functionality of the Zyla.
>
> Gerhard indeed gave a good synopsis of what real time processing goes on
> in sCMOS cameras, in order to turn raw images into acceptable images. In
> theory its possible to crack in and switch stuff like this off, but in most
> cases its not recommended. For most part, they are implemented in order
> to bring us to the situation we are used to with CCDs, except with lower
> noise, faster speed and wider dynamic range of course! That is to say, the
> processing is in place in order to provide us with a nice stable and uniform
> bias offset, with low read noise sitting on top, and uniform responsivity to
> light across the array.
>
> The most fundamental normalisation we do is to patch up the fixed pattern
> noise in the bias (baseline) offset. A comparison with and without is shown
> on P20 of the Andor sCMOS brochure:
>
>
>
> http://www.andor.com/pdfs/literature/Andor_sCMOS_Brochure.pdf
>
>
>
> Then there's a further round of real time bias correction called Dynamic
> Baseline Clamp.
>
> Gerhard also mentioned gain compensation. This is indeed implemented to
> normalise for slight responsivity differences between sCMOS pixels. A
> further side effect of not implementing this compensation would be to
> discernibly identify the two halves of the sensor when the sensor is
> illuminated.
>
>
>
> In our experience, the one area of real time processing that can represent
> a problem to people's usage, especially in pointillist super-resolution, is
> interpolative pixel filtering (referred to by Gerhard in context of hot pixels).
> In fact, Andor have now provided a means to switch off all interpolative
> filtering, such that no pixel values are replaced. Furthermore, if useful,
> we'll work with you to provide maps of where problematic pixels are
> located, such that at least you are aware of them. We are close to releasing
> a technical note on this to provide further detail and instruction. If you are
> interested, please contact my colleague Orla Hanrahan
> ([hidden email]) for specific detail.
>
>
>
> Best,
>
> Colin
>
>
>
> Dr Colin Coates
>
> Product Manager
>
> Andor Technology
>
>
>
>
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/

[Gerhard Holst]

*****
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*****

Hi Sergey,

in this perspective the onboard processing improves the linearity. I know from the earlier days of microscopy cameras that people are used to control the gain of a camera to get better, brighter images. If the so-called intra-scene dynamic of an image sensor is for example 1:4096 and the A/D-converter is able to resolve 12 bit, then additional gain just gives you a "white" image at a lower signal level (reducing the usable intra-scene dynamic).

********* Colin would call this the commercial part
In sCMOS with the different gain setting (if available) you can reduce the amount of data, but it does not increase the amount of information. Since the signal comes out simultaneously in two gain channels with 11 bit data, which are merged to deliver one 16 bit value (which covers the around 15 bit intra-scene dynamic of the camera depending on the corresponding data sheet of each manufacturer). How good this is done can always be measured with a photon transfer curve and a linearity measurement.
*********

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Sergey Tauger
Gesendet: Donnerstag, 15. Mai 2014 18:05
An: [hidden email]
Betreff: Re: AW: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

*****
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.
*****

Gerhard,

Thanks a lot for your reply. The main thing why I wanted to discard FPGA is that I
thought onboard processing makes images less linear. Now I think that my mistake
was choosing a wrong amplifier gain - I got really poor contrast.
http://www.andor.com/learning-academy/dual-amplifier-dynamic-range-scmos-
dynamic-range

Thanks,
Sergey

[Gerhard Holst]

Hi Zdenek,

I didn't intend to worry you, but I have not seen any CMOS image sensor with a raw, un-pre-processed, linear response.

During the FOM 2014 together with K. Bennett from Hamamatsu I gave a tutorial about digital cameras. On page 91
(http://www.pco.de/fileadmin/user_upload/20140412_FOM_CameraTutorial_PCO_fin_pdf.pdf) there is an example of a different CMIS image sensor showing the linearity before and after pre-processing. In CMOS image sensor the pure response is always more or less bended, but it can be linearized.

If you are interested in the behavior of the CIS2051 (which is now called CIS2521, a naming change) you might have a look to p 38 in the same document above, which shows a PTC curve and a linearity curve of a CIS2521 camera, measured by an independent EMVA1288 test lab. You will see, that the signal is very linear but the noise is not at all signal levels.

(Sorry for the link to our website, but I promised to the people who attended the tutorial to send them a pdf version, which was too large as an attachment, therefore I decided to provide in the publication section of our website).

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Zdenek Svindrych
Gesendet: Donnerstag, 15. Mai 2014 14:26
An: [hidden email]
Betreff: Re: AW: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

*****
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 Gerhard,



the linearization step surprised me a bit. When we do e.g. a STORM image
analysis, we work with Poisson statistics. This only holds when the camera's
physical response is linear. Can you tell how severe the nonlinear effects
are? At what intensities? I'm particularly interested in CIS2051 based
cameras...




Thanks,

zdenek svindrych

1st faculty of medicine, Charles University






---------- Původní zpráva ----------
Od: Gerhard Holst <[hidden email]>
Komu: [hidden email]
Datum: 15. 5. 2014 8:38:56
Předmět: AW: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 /
Flash4 V2)?

"*****
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 Sergey,

I don't want to hairsplit, and I can't answer this about the Zyla, because
it is not our camera, but the FPGA does usually all the control, timing,
readout of the images and the pre-processing. This pre-processing involves
some treatment of the raw data, that should be there to make useful images:
1. Fixed Pattern correction in the dark (=> offset correction, since every
column, pixel is a little bit different in offset)
2. Fixed Pattern correction in the bright (=> gain correction, since every
column, pixel is a little bit different in gain)
3. Linearization (the raw relationship between incoming photons and
outcoming signal is not linear)
4. Hot (dead) Pixel correction, (Usually a list with the position is stored
and the pixel value is replaced by a more or less weighted average of the
neighbor pixel, this is done in every digital camera), this is something
this can be switched off, if required, in many scientific cameras by a
certain command, as well in many cameras, this can be read out (keep in
mind, there doesn't exist an image sensor without such dead or hot pixels,
they are called "hot" because many of them appear with the maximum value in
the image, => white = hot pixels)
5. in the sCMOS cameras the manufacturers apply as well a dynamic algorithm
to do pretty much the same for the "blinking" pixels, since they appear like
"hot" or "warm" pixel, but not in every image, this filter usually can be
switched on and off by command.

Now, it is up to your application, what you want to switch off.

with best regards,

Gerhard
_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im
Auftrag von Sergey Tauger
Gesendet: Mittwoch, 14. Mai 2014 17:40
An: [hidden email]
Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 /
Flash4 V2)?

*****
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.
*****

Is it possible to turn off Zyla's onboar FPGA and compare data obtained from
the
same sample with and without onboard processing?

Sergey Tauger
MSU"

[Gerhard Holst]

*****
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 George,

but it is done on pixel level, as far as I know from the existing sCMOS cameras. What I understood from discussions with Dr. Bewersdorf, he had switched of the dynamic "blinker" filter in his cameras (a neighborhood operation), but he did not switch off the "hot" pixel correction (which is also a neighborhood operation, but always is on). As I mentioned both of the operations usually can be switched off (thanks to Colin for the confirmation, that Andor can do it, if it is of interest, our cameras can do this as well, and I think the Orcas too). But as far as I have seen the papers, if and how much it interferes has to be measured by doing reference experiments with or without the neighborhood filters on or off. Especially in localization microscopy Dr. Bewersdorf has shown, and I have seen similar results in a research project where we partnered, that sCMOS works nicely and improves the time consumption.

On the other hand, it is always possible to improve with more calculation power on a GPU, and I guess that's what the system manufacturer of localization microscopes do with all their specific experience of their methods and how they evaluate to achieve their PALM, STORM, dSTORM, GSD or whatsoever.

with best regards,

Gerhard

_______________________________

Dr. Gerhard Holst
Science & Research
PCO AG
Donaupark 11
93309 Kelheim, Germany
fon [phone]
fax [phone]
mob [phone]
[hidden email]
www.pco.de


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von George McNamara
Gesendet: Freitag, 16. Mai 2014 01:20
An: [hidden email]
Betreff: Re: AW: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?

*****
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 Colin,

Could you (and Gerhard/PCO and someone from Hamamatsu) please explain
why you are not correcting the gain for every pixel, re: the mess shown
in Huang ... Bewersdorf et al 2013,
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696415/ , supplemental
figure 1,

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696415/bin/NIHMS475969-supplement-1.pdf

This should be doable at the camera device driver level (would be nice
if implemented 'realtime' on a GPU or Intel Phi card) so that everyone
who owns any of your cameras could get this for just the effort of a
driver update.

thanks in advance,

George

On 5/15/2014 11:46 AM, Colin Coates 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.
> *****
>
> **Commercial response**
>
> Hi,
>
> Just to chip in on the question of manipulating the FPGA processing
> functionality of the Zyla.
>
> Gerhard indeed gave a good synopsis of what real time processing goes on
> in sCMOS cameras, in order to turn raw images into acceptable images. In
> theory its possible to crack in and switch stuff like this off, but in most
> cases its not recommended. For most part, they are implemented in order
> to bring us to the situation we are used to with CCDs, except with lower
> noise, faster speed and wider dynamic range of course! That is to say, the
> processing is in place in order to provide us with a nice stable and uniform
> bias offset, with low read noise sitting on top, and uniform responsivity to
> light across the array.
>
> The most fundamental normalisation we do is to patch up the fixed pattern
> noise in the bias (baseline) offset. A comparison with and without is shown
> on P20 of the Andor sCMOS brochure:
>
>
>
> http://www.andor.com/pdfs/literature/Andor_sCMOS_Brochure.pdf
>
>
>
> Then there's a further round of real time bias correction called Dynamic
> Baseline Clamp.
>
> Gerhard also mentioned gain compensation. This is indeed implemented to
> normalise for slight responsivity differences between sCMOS pixels. A
> further side effect of not implementing this compensation would be to
> discernibly identify the two halves of the sensor when the sensor is
> illuminated.
>
>
>
> In our experience, the one area of real time processing that can represent
> a problem to people's usage, especially in pointillist super-resolution, is
> interpolative pixel filtering (referred to by Gerhard in context of hot pixels).
> In fact, Andor have now provided a means to switch off all interpolative
> filtering, such that no pixel values are replaced. Furthermore, if useful,
> we'll work with you to provide maps of where problematic pixels are
> located, such that at least you are aware of them. We are close to releasing
> a technical note on this to provide further detail and instruction. If you are
> interested, please contact my colleague Orla Hanrahan
> ([hidden email]) for specific detail.
>
>
>
> Best,
>
> Colin
>
>
>
> Dr Colin Coates
>
> Product Manager
>
> Andor Technology
>
>
>
>
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/

[George McNamara]

*****
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 Gerhard,

Your FOM 2014 presentation

http://www.pco.de/fileadmin/user_upload/20140412_FOM_CameraTutorial_PCO_fin_pdf.pdf

is titled "part 1, and the last page (p. 103) has "end of part I". Do
you have a web link for part 2 (or is that Keith Bennett's presentation?).

Thanks for your comments about this. You state on page 93 that it is
important to include the offset,

"to allow the access of the complete readout noise."

but, really, none of my users in Los Angeles (5 years), Miami (6 years)
or colleagues in Houston (1+ years), knew how to interpret the 'offset'
image data, and more importantly did not care. We can always see what
the camera is outputting by blocking any light from reaching the camera
and taking 'dark reference' images (plural, per Borisy's law: never rely
on N=1).

I suggest that for most light microscopy users, it would be better for
the image data reaching the application and user be linear with respect
to photons in each pixel and no photons give zero intensity to the user.
Best default output would be 1 photoelectron --> 1 intensity level. Of
course I also want this with instant gratification (and zero or not too
much more expense) which brings me to cleaning up the output:

With respect to speeding up calculations to get the best data to the
application and user, in addition to the NVidia TITAN Z GPU , 8
Teraflops, 6 Gb ram, $3000 - and the "Z" is perfect moniker for
deconvolving and otherwise processing microscope Z-series - there is
also the upcoming Intel next gen Phi "Knights Landing" (KNL) CPU,
initially 3 Teraflops per CPU (full instruction set of a CPU), access to
a whole lot of ram, no price info (release scheduled 2015), see
http://vr-zone.com/articles/xeon-phi-knights-series-continues-landing-2015/64112.html

Your page 100 mentions 10 Gigabit Ethernet - the vr-zone article above
mentions 100 Gigabit interconnect the KNL will use. Maybe your cameras
should go to this or the 56 Gigabit interface ... or talk to NVidia to
enable a bridge direct between your digitizer card and the TITAN Z.

George



On 5/16/2014 1:54 AM, Gerhard Holst wrote:

> Hi Zdenek,
>
> I didn't intend to worry you, but I have not seen any CMOS image sensor with a raw, un-pre-processed, linear response.
>
> During the FOM 2014 together with K. Bennett from Hamamatsu I gave a tutorial about digital cameras. On page 91
> (http://www.pco.de/fileadmin/user_upload/20140412_FOM_CameraTutorial_PCO_fin_pdf.pdf) there is an example of a different CMIS image sensor showing the linearity before and after pre-processing. In CMOS image sensor the pure response is always more or less bended, but it can be linearized.
>
> If you are interested in the behavior of the CIS2051 (which is now called CIS2521, a naming change) you might have a look to p 38 in the same document above, which shows a PTC curve and a linearity curve of a CIS2521 camera, measured by an independent EMVA1288 test lab. You will see, that the signal is very linear but the noise is not at all signal levels.
>
> (Sorry for the link to our website, but I promised to the people who attended the tutorial to send them a pdf version, which was too large as an attachment, therefore I decided to provide in the publication section of our website).
>
> with best regards,
>
> Gerhard
> _______________________________
>
> Dr. Gerhard Holst
> Science&  Research
> PCO AG
> Donaupark 11
> 93309 Kelheim, Germany
> fon [phone]
> fax [phone]
> mob [phone]
> [hidden email]
> www.pco.de
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Zdenek Svindrych
> Gesendet: Donnerstag, 15. Mai 2014 14:26
> An: [hidden email]
> Betreff: Re: AW: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 / Flash4 V2)?
>
> *****
> 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 Gerhard,
>
>
>
> the linearization step surprised me a bit. When we do e.g. a STORM image
> analysis, we work with Poisson statistics. This only holds when the camera's
> physical response is linear. Can you tell how severe the nonlinear effects
> are? At what intensities? I'm particularly interested in CIS2051 based
> cameras...
>
>
>
>
> Thanks,
>
> zdenek svindrych
>
> 1st faculty of medicine, Charles University
>
>
>
>
>
>
> ---------- Původní zpráva ----------
> Od: Gerhard Holst<[hidden email]>
> Komu: [hidden email]
> Datum: 15. 5. 2014 8:38:56
> Předmět: AW: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 /
> Flash4 V2)?
>
> "*****
> 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 Sergey,
>
> I don't want to hairsplit, and I can't answer this about the Zyla, because
> it is not our camera, but the FPGA does usually all the control, timing,
> readout of the images and the pre-processing. This pre-processing involves
> some treatment of the raw data, that should be there to make useful images:
> 1. Fixed Pattern correction in the dark (=>  offset correction, since every
> column, pixel is a little bit different in offset)
> 2. Fixed Pattern correction in the bright (=>  gain correction, since every
> column, pixel is a little bit different in gain)
> 3. Linearization (the raw relationship between incoming photons and
> outcoming signal is not linear)
> 4. Hot (dead) Pixel correction, (Usually a list with the position is stored
> and the pixel value is replaced by a more or less weighted average of the
> neighbor pixel, this is done in every digital camera), this is something
> this can be switched off, if required, in many scientific cameras by a
> certain command, as well in many cameras, this can be read out (keep in
> mind, there doesn't exist an image sensor without such dead or hot pixels,
> they are called "hot" because many of them appear with the maximum value in
> the image, =>  white = hot pixels)
> 5. in the sCMOS cameras the manufacturers apply as well a dynamic algorithm
> to do pretty much the same for the "blinking" pixels, since they appear like
> "hot" or "warm" pixel, but not in every image, this filter usually can be
> switched on and off by command.
>
> Now, it is up to your application, what you want to switch off.
>
> with best regards,
>
> Gerhard
> _______________________________
>
> Dr. Gerhard Holst
> Science&  Research
> PCO AG
> Donaupark 11
> 93309 Kelheim, Germany
> fon [phone]
> fax [phone]
> mob [phone]
> [hidden email]
> www.pco.de
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]] Im
> Auftrag von Sergey Tauger
> Gesendet: Mittwoch, 14. Mai 2014 17:40
> An: [hidden email]
> Betreff: Re: Anyone compared recently released sCMOS? (PCO 4.2 / Zyla 4.2 /
> Flash4 V2)?
>
> *****
> 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.
> *****
>
> Is it possible to turn off Zyla's onboar FPGA and compare data obtained from
> the
> same sample with and without onboard processing?
>
> Sergey Tauger
> MSU"
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/