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back illuminates sCMOS vs emCCD

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Jeff Spector

back illuminates sCMOS vs emCCD

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Greetings,
We're thinking about replacing some ~ 10 year old iXON 897's on a single
molecule two color,two camera TIRF system. I've seen some comparisons
between the earlier scmos cameras and emCCDs. I would like to get the
opinions of people on this list as to which camera would be the best for
single molecule imaging. The single gfp's we image by. TIRF will be
diffusing and not stationary. A lot of the comparisons are for
localization microscopy where the target is stationary. We are
particularly interested in real world examples of diffusing single
fluorophores. Any experience or thoughts are appreciated.
Thanks,
-Jeff

Krishna Mudumbi-2

Re: back illuminates sCMOS vs emCCD

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Hi Jeff,

I tested the Andor iXon-897 EM-CCD, and the Prime 95B both specifically for
single-particle tracking (SPT). I think they both performed rather well and
I honestly don't think you can go very wrong with either (others may feel
differently), but your conditions may make you lean one way or another. If
field of view is important to you, I would recommend the Prime 95B, if,
however, like me you wanted some extra sensitivity when you have weak
fluors (smFRET in my case), then the Andor EM-CCD might be your best bet.

Cheers,

Krishna

-------------------------
Krishna C. Mudumbi, Ph.D.
NCI Outstanding Early Stage K99 Fellow
Department of Pharmacology | Yale Cancer Biology Institute
Advanced Biosciences Center, Room 311
840 West Campus Drive
West Haven, CT 06516
Email: [hidden email]

On Mon, Feb 15, 2021, 5:17 PM Jeff Spector <[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.
> *****
>
> Greetings,
> We're thinking about replacing some ~ 10 year old iXON 897's on a single
> molecule two color,two camera TIRF system. I've seen some comparisons
> between the earlier scmos cameras and emCCDs. I would like to get the
> opinions of people on this list as to which camera would be the best for
> single molecule imaging. The single gfp's we image by. TIRF will be
> diffusing and not stationary. A lot of the comparisons are for
> localization microscopy where the target is stationary. We are
> particularly interested in real world examples of diffusing single
> fluorophores. Any experience or thoughts are appreciated.
> Thanks,
> -Jeff
>

Tim Feinstein

Re: back illuminates sCMOS vs emCCD

In reply to this post by Jeff Spector

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Hi Jeff,

As a 95b user I confirm that sCMOS is an excellent choice. However if you use a sCMOS for single molecule localization then I suggest you keep in mind that traditional single-molecule protocols assume a uniformity of noise that you find in CCD and EMCCD chips but is not true for sCMOS chips. Make sure you consult the vendor and check the literature on this, for example Mandracchia et al. (2020), Nature Comms 11: #94, to minimize artifacts with sCMOS. I believe the magnitude of the non-uniformity is less of a concern for most users, but single molecule tracking demands a lot from a chip.

All the best,

T

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

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jeff Spector
Sent: Monday, February 15, 2021 4:49 PM
To: [hidden email]
Subject: back illuminates sCMOS vs emCCD

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Greetings,
We're thinking about replacing some ~ 10 year old iXON 897's on a single molecule two color,two camera TIRF system. I've seen some comparisons between the earlier scmos cameras and emCCDs. I would like to get the opinions of people on this list as to which camera would be the best for single molecule imaging. The single gfp's we image by. TIRF will be diffusing and not stationary. A lot of the comparisons are for localization microscopy where the target is stationary. We are particularly interested in real world examples of diffusing single fluorophores. Any experience or thoughts are appreciated.
Thanks,
-Jeff

RJ3

Re: back illuminates sCMOS vs emCCD

In reply to this post by Jeff Spector

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There is nothing quite like the EMCCD 897 so I think you will be disappointed to find an alternative.
What is your motivation? More gain? Not going to find it.

More resolution or higher speed? These are possible.

Look beyond the max QE. The response curves are different and you will also miss the EM gain. The enormous pixels of the 897 hurt the spatial resolution but help its sensitivity, take caution if you have to software bin to get things where you need on cameras with smaller pixels.

Jeff Spector

Re: back illuminates sCMOS vs emCCD

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Hi,
Thanks for all the advice so far. Please keep it coming. To answer a few
questions :

On Tue, Feb 16, 2021 at 10:43 AM RJ <[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.
> *****
>
> There is nothing quite like the EMCCD 897 so I think you will be
> disappointed to find an alternative.
> What is your motivation? More gain? Not going to find it.
>
> More resolution or higher speed? These are possible.
>
> Look beyond the max QE. The response curves are different and you will
> also miss the EM gain. The enormous pixels of the 897 hurt the spatial
> resolution but help its sensitivity, take caution if you have to software
> bin to get things where you need on cameras with smaller pixels.
>

We were drawn to the sCMOS mainly because of the large (very large
compared to an 897) field of view. Yes, the sCMOS isn't as sensitive as
emCCD in the super low photon/pixel limit ( at least on paper), which is
why I've reached out to get some real world opinions. We currently don't
use any binning on the emCCD. I should mention out emCCD's are ~ 10 years
old so I"m sure they aren't as sensitive as they used to be. It sounds like
we would be ok replacing an emccd with the scmos, but I'd still like to
hear from someone who has done single fluorophore tracking with an sCMOS
and how it compared to emCCD.

- Jeff

Gerhard Holst

AW: back illuminates sCMOS vs emCCD

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Hi Jeff,

the emCCDs with their inbuild gain are nice and also many optical systems for microscopes were optimized for the large pixel sizes. You use sensitive as term to describe how good your emCCDs react to light. In case they are old, and continuously used, I would be more worried about their gain, because the nice effect of impact ionization is as well continuously destroying the semiconductor lattice, that's way the gain curves in emCCD cameras are changing with aging.

Maybe, for your existing typical application you could do some rough back-calculations to estimate how many photons you usually "see". As rule of thumb I would say if you signal per pixel is more than 10 photons, then sCMOS can be a replacement. Below that still the gain of the emCCDs has an advantage.

In terms of quantum efficiency (if you call that the "sensitivity") of the image sensor, there is no difference, maybe the spectral position of the maximum might be slightly different, but for both types of image sensors you will find versions with 80 - 95% QE (which are best possible measured with +/- 5%), so the sensitivity of the image detector is not the major issue.

What can be more critical is your optical situation, which corresponds to what RJ has written. If you want a plugin replacement for your emCCD camera, you would need a camera with the same pixel size. If you replace the existing camera with for example 11um pixel size by a camera with 6.5 um pixel size you will roughly get a 1/4 of the signal just by geometry and not sensitivity. If you want to use such a camera, you will need to reduce your illumination circle to increase the "photon density". Then you could exploit the lower readout noise and higher speed of the sCMOS cameras.

I am not going to repeat all the comparisons of advantages / disadvantages between emCCD and sCMOS, there is a lot of well written literature out there, at the end you need the best possible camera for your measurements.

Regards,
Gerhard

Dr. Gerhard Holst
Head of Research & Science
Business Development
+49 (0) 9441 2005 570

PCO AG, Donaupark 11, 93309 Kelheim, Germany, www.pco.de
Vorstand / Managing Board: Alexander Grünig, Luitpold Kaspar
Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Dr. Emil Ott
USt. ID-Nr. / VAT: DE128590843, Registergericht / Register Court: Amtsgericht Regensburg HRB 9157
Sitz der Gesellschaft / Registered Office: Kelheim

-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List <[hidden email]> Im Auftrag von Jeff Spector
Gesendet: Dienstag, 16. Februar 2021 19:38
An: [hidden email]
Betreff: Re: back illuminates sCMOS vs emCCD

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*****

Hi,
Thanks for all the advice so far. Please keep it coming. To answer a few questions :

On Tue, Feb 16, 2021 at 10:43 AM RJ <[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.
> *****
>
> There is nothing quite like the EMCCD 897 so I think you will be
> disappointed to find an alternative.
> What is your motivation? More gain? Not going to find it.
>
> More resolution or higher speed? These are possible.
>
> Look beyond the max QE. The response curves are different and you will
> also miss the EM gain. The enormous pixels of the 897 hurt the spatial
> resolution but help its sensitivity, take caution if you have to
> software bin to get things where you need on cameras with smaller pixels.
>

We were drawn to the sCMOS mainly because of the large (very large compared to an 897) field of view. Yes, the sCMOS isn't as sensitive as emCCD in the super low photon/pixel limit ( at least on paper), which is why I've reached out to get some real world opinions. We currently don't use any binning on the emCCD. I should mention out emCCD's are ~ 10 years old so I"m sure they aren't as sensitive as they used to be. It sounds like we would be ok replacing an emccd with the scmos, but I'd still like to hear from someone who has done single fluorophore tracking with an sCMOS and how it compared to emCCD.

- Jeff

Alan Mullan

Re: AW: back illuminates sCMOS vs emCCD

In reply to this post by Jeff Spector

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Hi Jeff,
I thought I'd wait for the users of the various cameras to comment on their experiences. From our side as manufacturers of the iXon 897 models you have currently, the latest EMCCD models and back-illuminated sCMOS cameras we can make some comments from what we see.

As others have mentioned it comes down to your imaging priorities and optical matching that Gerhard already described. The iXon 897, although now an older EMCCD model should perform very well - that model did have our RealGain and EMCal features that helped avoid issues with EM gain ageing. I would doubt there would be any issues from EM ageing affecting performance, and if it still cools as well as it did then we see these models continue to give comparable performance to when new. The newer Ultra 897 EMCCD models of course have been updated and have revised electronics for lower internal noise, which helps with very low signals and most notably the increased speeds.

The EMCCD cameras still give the best sensitivity so when you need that there is no equal. To maintain similar sensitivity, but improve the field of view an option is the Ultra 888 model (or other EMCCD models that use the larger 1024x1024 sensor), that gives a much larger sensor area the same size as the 4.2 megapixel sCMOS format albeit using a 13 µm pixel size. We find many customers with the older iXon models often go that route over back-illuminated sCMOS when demoing for single molecule experiments due to the hit in sensitivity they would take.

For the back-illuminated sCMOS side, the main sensor generally in this kind of application being the GS400BSI- our camera model with this sensor is the Sona 4.2B-11. If you have enough photons with what you are working with, you can potentially get up to 32 mm field of view (and higher speeds). So it can offer a big benefit for some.

Testing is always recommended against your current cameras if it suits as there are too many subtleties in parameters from labels to optics involved.

Michael Giacomelli-2

Re: [EXT] AW: back illuminates sCMOS vs emCCD

In reply to this post by Gerhard Holst

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Hi Gerhard,

Care is needed when comparing QE figures given the differences between how
these sensors operate. An EMCCD has internal gain, which means it has
excess noise (similar to a PMT) . The effective QE (that is, the QE you
would calculate if you measured your shot noise for a known number of
photons) is lower than the nominal QE because the gain process introduces
additional shot noise. I have no personal experience with these sensors,
but Hamamatsu specs an excess noise factor of (1.4)^2 for their EMCCDs.
Thus the effective QE would be divided by that factor, giving ~95% with EM
off, but only ~ 50% with EM on.

I saw Fairchild has announced new sCMOS sensors that obtain sub-electron
read noise. It does not seem like they're shipping these yet, but I think
eventually these will replace EMCCD since they should have similar low
light sensitivity to EMCCD without the excess noise.

Mike

On Wed, Feb 17, 2021 at 2:35 AM Gerhard Holst <[hidden email]> wrote:

Gerhard Holst

AW: [EXT] AW: back illuminates sCMOS vs emCCD

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Hi Mike,

As far as I know the QE curves in emCCDs are specified with gain = 1, because usually the quantum efficiency refers to the pixels which detect the light and not the gain registers. I absolutely agree, that the additional noise factors have to be taken into account, but the gain doesn't reduce the quantum efficiency, but it reduces the usable intra-scene dynamic range. The higher readout noise of the emCCDs is more than compensated by the gain before readout.

In my understanding, the quantum efficiency of such a detector (assuming a linear detector model, and assuming that we are above 250nm, such that one photon can generate one charge carrier at max) how many charge carriers, let's say electrons, I get from so many photons hitting the detector.

Best,
Gerhard

Dr. Gerhard Holst
Head of Research & Science
Business Development
+49 (0) 9441 2005 570

PCO AG, Donaupark 11, 93309 Kelheim, Germany, www.pco.de
Vorstand / Managing Board: Alexander Grünig, Luitpold Kaspar
Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Dr. Emil Ott
USt. ID-Nr. / VAT: DE128590843, Registergericht / Register Court: Amtsgericht Regensburg HRB 9157
Sitz der Gesellschaft / Registered Office: Kelheim

-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List <[hidden email]> Im Auftrag von Michael Giacomelli
Gesendet: Mittwoch, 17. Februar 2021 16:48
An: [hidden email]
Betreff: Re: [EXT] AW: back illuminates sCMOS vs emCCD

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Hi Gerhard,

Care is needed when comparing QE figures given the differences between how these sensors operate. An EMCCD has internal gain, which means it has excess noise (similar to a PMT) . The effective QE (that is, the QE you would calculate if you measured your shot noise for a known number of
photons) is lower than the nominal QE because the gain process introduces additional shot noise. I have no personal experience with these sensors, but Hamamatsu specs an excess noise factor of (1.4)^2 for their EMCCDs.
Thus the effective QE would be divided by that factor, giving ~95% with EM off, but only ~ 50% with EM on.

I saw Fairchild has announced new sCMOS sensors that obtain sub-electron read noise. It does not seem like they're shipping these yet, but I think eventually these will replace EMCCD since they should have similar low light sensitivity to EMCCD without the excess noise.

Mike

On Wed, Feb 17, 2021 at 2:35 AM Gerhard Holst <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
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> and include the link in your posting.
> *****
>
> Hi Jeff,
>
> the emCCDs with their inbuild gain are nice and also many optical
> systems for microscopes were optimized for the large pixel sizes. You
> use sensitive as term to describe how good your emCCDs react to light.
> In case they are old, and continuously used, I would be more worried
> about their gain, because the nice effect of impact ionization is as
> well continuously destroying the semiconductor lattice, that's way the
> gain curves in emCCD cameras are changing with aging.
>
> Maybe, for your existing typical application you could do some rough
> back-calculations to estimate how many photons you usually "see". As
> rule of thumb I would say if you signal per pixel is more than 10
> photons, then sCMOS can be a replacement. Below that still the gain of
> the emCCDs has an advantage.
>
> In terms of quantum efficiency (if you call that the "sensitivity") of
> the image sensor, there is no difference, maybe the spectral position
> of the maximum might be slightly different, but for both types of
> image sensors you will find versions with 80 - 95% QE (which are best
> possible measured with +/- 5%), so the sensitivity of the image
> detector is not the major issue.
>
> What can be more critical is your optical situation, which corresponds
> to what RJ has written. If you want a plugin replacement for your
> emCCD camera, you would need a camera with the same pixel size. If you
> replace the existing camera with for example 11um pixel size by a
> camera with 6.5 um pixel size you will roughly get a 1/4 of the signal
> just by geometry and not sensitivity. If you want to use such a
> camera, you will need to reduce your illumination circle to increase
> the "photon density". Then you could exploit the lower readout noise and higher speed of the sCMOS cameras.
>
> I am not going to repeat all the comparisons of advantages /
> disadvantages between emCCD and sCMOS, there is a lot of well written
> literature out there, at the end you need the best possible camera for your measurements.
>
> Regards,
> Gerhard
>
>
>
>
> Dr. Gerhard Holst
> Head of Research & Science
> Business Development
> +49 (0) 9441 2005 570
>
> PCO AG, Donaupark 11, 93309 Kelheim, Germany,
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.pco.de&d=DwIGa
> Q&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOI
> XyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&
> s=8ySLjREt6J_XNvS7vfaDK_Jt3OJT8YGQHmE8-cDsdg0&e=
> Vorstand / Managing Board: Alexander Grünig, Luitpold Kaspar
> Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Dr.
> Emil Ott
> USt. ID-Nr. / VAT: DE128590843, Registergericht / Register Court:
> Amtsgericht Regensburg HRB 9157
> Sitz der Gesellschaft / Registered Office: Kelheim
>
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List <[hidden email]> Im
> Auftrag von Jeff Spector
> Gesendet: Dienstag, 16. Februar 2021 19:38
> An: [hidden email]
> Betreff: Re: back illuminates sCMOS vs emCCD
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> JVnnxrlDLNPkGMfko&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
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> EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> and include the link in your posting.
> *****
>
> Hi,
> Thanks for all the advice so far. Please keep it coming. To answer a
> few questions :
>
> On Tue, Feb 16, 2021 at 10:43 AM RJ <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=qG
> msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> JVnnxrlDLNPkGMfko&e=
> > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> eOIXyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPa
> EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> and include the link in your posting.
> > *****
> >
> > There is nothing quite like the EMCCD 897 so I think you will be
> > disappointed to find an alternative.
> > What is your motivation? More gain? Not going to find it.
> >
> > More resolution or higher speed? These are possible.
> >
> > Look beyond the max QE. The response curves are different and you
> > will also miss the EM gain. The enormous pixels of the 897 hurt the
> > spatial resolution but help its sensitivity, take caution if you
> > have to software bin to get things where you need on cameras with smaller pixels.
> >
>
> We were drawn to the sCMOS mainly because of the large (very large
> compared to an 897) field of view. Yes, the sCMOS isn't as sensitive
> as emCCD in the super low photon/pixel limit ( at least on paper),
> which is why I've reached out to get some real world opinions. We
> currently don't use any binning on the emCCD. I should mention out
> emCCD's are ~ 10 years old so I"m sure they aren't as sensitive as
> they used to be. It sounds like we would be ok replacing an emccd with
> the scmos, but I'd still like to hear from someone who has done single
> fluorophore tracking with an sCMOS and how it compared to emCCD.
>
> - Jeff
>

Giang, William-2

Re: [EXT] AW: back illuminates sCMOS vs emCCD

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Mike did specify *effective* QE but should have mentioned this is in the context of SNR calculations.

IMO Hamamatsu explains it best in their Flash4 white paper at https://www.hamamatsu.com/resources/pdf/sys/e_flash4_whitepaper.pdf

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Gerhard Holst <[hidden email]>
Sent: Wednesday, February 17, 2021 11:09:27 AM
To: [hidden email]
Subject: AW: [EXT] AW: back illuminates sCMOS vs emCCD

Attention: This email originated outside of Penn State Health. Use caution when clicking links or opening attachments.

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Hi Mike,

As far as I know the QE curves in emCCDs are specified with gain = 1, because usually the quantum efficiency refers to the pixels which detect the light and not the gain registers. I absolutely agree, that the additional noise factors have to be taken into account, but the gain doesn't reduce the quantum efficiency, but it reduces the usable intra-scene dynamic range. The higher readout noise of the emCCDs is more than compensated by the gain before readout.

In my understanding, the quantum efficiency of such a detector (assuming a linear detector model, and assuming that we are above 250nm, such that one photon can generate one charge carrier at max) how many charge carriers, let's say electrons, I get from so many photons hitting the detector.

Best,
Gerhard

Dr. Gerhard Holst
Head of Research & Science
Business Development
+49 (0) 9441 2005 570

PCO AG, Donaupark 11, 93309 Kelheim, Germany, https://urldefense.com/v3/__http://www.pco.de__;!!Ls64Rlj6!l4izPqMnkZnxrEDc3UqxyzLrAQfG12VjfifWRj9X4HtFjrfMP-j1NKeIln547-B8Ku8Nh_E$
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-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List <[hidden email]> Im Auftrag von Michael Giacomelli
Gesendet: Mittwoch, 17. Februar 2021 16:48
An: [hidden email]
Betreff: Re: [EXT] AW: back illuminates sCMOS vs emCCD

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Hi Gerhard,

Care is needed when comparing QE figures given the differences between how these sensors operate. An EMCCD has internal gain, which means it has excess noise (similar to a PMT) . The effective QE (that is, the QE you would calculate if you measured your shot noise for a known number of
photons) is lower than the nominal QE because the gain process introduces additional shot noise. I have no personal experience with these sensors, but Hamamatsu specs an excess noise factor of (1.4)^2 for their EMCCDs.
Thus the effective QE would be divided by that factor, giving ~95% with EM off, but only ~ 50% with EM on.

I saw Fairchild has announced new sCMOS sensors that obtain sub-electron read noise. It does not seem like they're shipping these yet, but I think eventually these will replace EMCCD since they should have similar low light sensitivity to EMCCD without the excess noise.

Mike

On Wed, Feb 17, 2021 at 2:35 AM Gerhard Holst <[hidden email]> wrote:

Michael Giacomelli-2

Re: [EXT] AW: back illuminates sCMOS vs emCCD

In reply to this post by Gerhard Holst

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Hi Gerhard,

Gain doesn't reduce the quantum efficiency of the pixel, rather it reduces
the effective quantum efficiency from the point of view of the user, that
is the QE you would calculate if you measured your shot noise for a known
number of photons. For example, suppose your sensor has a QE of 50% and an
excess noise factor of 1. If you illuminate it with 10 photons per pixel,
you would measure a shot noise limited snr of sqrt(5) and conclude that the
QE is 50%. Similarly for a detector with a 100% nominal QE with an ENF of
2, 10 photons would also give you a sqrt(5), leading you to again measure a
50% QE. This measurement is wrong from the point of view of the pixel (it
detects every photon), but from a user perspective looking at the whole
system, the sensor functions as if the QE is 50%. An alternative way to
think about it is that while the pixel is converting every photon into a
photoelectron, the stochastic nature of the electron multiplication process
results in some of those photoelectrons failing to contribute to your SNR.

This is similar to the situation with PMTs, where the QE of the
photocathode is specified in the datasheet, but if you measure shot noise,
you'll typically calculate that the QE is about half the value in the
datasheet due to various losses and noise sources in the electron
multiplication process (after the photocathode).

Mike

On Wed, Feb 17, 2021 at 11:52 AM Gerhard Holst <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> Post images on
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> and include the link in your posting.
> *****
>
> Hi Mike,
>
> As far as I know the QE curves in emCCDs are specified with gain = 1,
> because usually the quantum efficiency refers to the pixels which detect
> the light and not the gain registers. I absolutely agree, that the
> additional noise factors have to be taken into account, but the gain
> doesn't reduce the quantum efficiency, but it reduces the usable
> intra-scene dynamic range. The higher readout noise of the emCCDs is more
> than compensated by the gain before readout.
>
> In my understanding, the quantum efficiency of such a detector (assuming a
> linear detector model, and assuming that we are above 250nm, such that one
> photon can generate one charge carrier at max) how many charge carriers,
> let's say electrons, I get from so many photons hitting the detector.
>
> Best,
> Gerhard
>
>
> Dr. Gerhard Holst
> Head of Research & Science
> Business Development
> +49 (0) 9441 2005 570
>
> PCO AG, Donaupark 11, 93309 Kelheim, Germany,
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.pco.de&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=WAvC5yKfqp8z6AtH8KOwB2qkOooJ8eF7PZdBFQSJbf0&s=lQdVIGx-B_6lGDhsrzDduA5xZHLQcpopKZfSCji6__8&e=
> Vorstand / Managing Board: Alexander Grünig, Luitpold Kaspar
> Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Dr.
> Emil Ott
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> Amtsgericht Regensburg HRB 9157
> Sitz der Gesellschaft / Registered Office: Kelheim
>
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List <[hidden email]> Im
> Auftrag von Michael Giacomelli
> Gesendet: Mittwoch, 17. Februar 2021 16:48
> An: [hidden email]
> Betreff: Re: [EXT] AW: back illuminates sCMOS vs emCCD
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=WAvC5yKfqp8z6AtH8KOwB2qkOooJ8eF7PZdBFQSJbf0&s=qf-L1Gc3xO1zA2ZFq_31JjzoUS6tSbUff_u7-adCl3Y&e=
> Post images on
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> and include the link in your posting.
> *****
>
> Hi Gerhard,
>
> Care is needed when comparing QE figures given the differences between how
> these sensors operate. An EMCCD has internal gain, which means it has
> excess noise (similar to a PMT) . The effective QE (that is, the QE you
> would calculate if you measured your shot noise for a known number of
> photons) is lower than the nominal QE because the gain process introduces
> additional shot noise. I have no personal experience with these sensors,
> but Hamamatsu specs an excess noise factor of (1.4)^2 for their EMCCDs.
> Thus the effective QE would be divided by that factor, giving ~95% with EM
> off, but only ~ 50% with EM on.
>
> I saw Fairchild has announced new sCMOS sensors that obtain sub-electron
> read noise. It does not seem like they're shipping these yet, but I think
> eventually these will replace EMCCD since they should have similar low
> light sensitivity to EMCCD without the excess noise.
>
> Mike
>
> On Wed, Feb 17, 2021 at 2:35 AM Gerhard Holst <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=qG
> > msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> > JVnnxrlDLNPkGMfko&e=
> > Post images on
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPa
> > EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> > and include the link in your posting.
> > *****
> >
> > Hi Jeff,
> >
> > the emCCDs with their inbuild gain are nice and also many optical
> > systems for microscopes were optimized for the large pixel sizes. You
> > use sensitive as term to describe how good your emCCDs react to light.
> > In case they are old, and continuously used, I would be more worried
> > about their gain, because the nice effect of impact ionization is as
> > well continuously destroying the semiconductor lattice, that's way the
> > gain curves in emCCD cameras are changing with aging.
> >
> > Maybe, for your existing typical application you could do some rough
> > back-calculations to estimate how many photons you usually "see". As
> > rule of thumb I would say if you signal per pixel is more than 10
> > photons, then sCMOS can be a replacement. Below that still the gain of
> > the emCCDs has an advantage.
> >
> > In terms of quantum efficiency (if you call that the "sensitivity") of
> > the image sensor, there is no difference, maybe the spectral position
> > of the maximum might be slightly different, but for both types of
> > image sensors you will find versions with 80 - 95% QE (which are best
> > possible measured with +/- 5%), so the sensitivity of the image
> > detector is not the major issue.
> >
> > What can be more critical is your optical situation, which corresponds
> > to what RJ has written. If you want a plugin replacement for your
> > emCCD camera, you would need a camera with the same pixel size. If you
> > replace the existing camera with for example 11um pixel size by a
> > camera with 6.5 um pixel size you will roughly get a 1/4 of the signal
> > just by geometry and not sensitivity. If you want to use such a
> > camera, you will need to reduce your illumination circle to increase
> > the "photon density". Then you could exploit the lower readout noise and
> higher speed of the sCMOS cameras.
> >
> > I am not going to repeat all the comparisons of advantages /
> > disadvantages between emCCD and sCMOS, there is a lot of well written
> > literature out there, at the end you need the best possible camera for
> your measurements.
> >
> > Regards,
> > Gerhard
> >
> >
> >
> >
> > Dr. Gerhard Holst
> > Head of Research & Science
> > Business Development
> > +49 (0) 9441 2005 570
> >
> > PCO AG, Donaupark 11, 93309 Kelheim, Germany,
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.pco.de&d=DwIGa
> > Q&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOI
> > XyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&
> > s=8ySLjREt6J_XNvS7vfaDK_Jt3OJT8YGQHmE8-cDsdg0&e=
> > Vorstand / Managing Board: Alexander Grünig, Luitpold Kaspar
> > Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Dr.
> > Emil Ott
> > USt. ID-Nr. / VAT: DE128590843, Registergericht / Register Court:
> > Amtsgericht Regensburg HRB 9157
> > Sitz der Gesellschaft / Registered Office: Kelheim
> >
> >
> >
> > -----Ursprüngliche Nachricht-----
> > Von: Confocal Microscopy List <[hidden email]> Im
> > Auftrag von Jeff Spector
> > Gesendet: Dienstag, 16. Februar 2021 19:38
> > An: [hidden email]
> > Betreff: Re: back illuminates sCMOS vs emCCD
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=qG
> > msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> > JVnnxrlDLNPkGMfko&e=
> > Post images on
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPa
> > EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> > and include the link in your posting.
> > *****
> >
> > Hi,
> > Thanks for all the advice so far. Please keep it coming. To answer a
> > few questions :
> >
> > On Tue, Feb 16, 2021 at 10:43 AM RJ <[hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=qG
> > msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> > JVnnxrlDLNPkGMfko&e=
> > > Post images on
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPa
> > EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> > and include the link in your posting.
> > > *****
> > >
> > > There is nothing quite like the EMCCD 897 so I think you will be
> > > disappointed to find an alternative.
> > > What is your motivation? More gain? Not going to find it.
> > >
> > > More resolution or higher speed? These are possible.
> > >
> > > Look beyond the max QE. The response curves are different and you
> > > will also miss the EM gain. The enormous pixels of the 897 hurt the
> > > spatial resolution but help its sensitivity, take caution if you
> > > have to software bin to get things where you need on cameras with
> smaller pixels.
> > >
> >
> > We were drawn to the sCMOS mainly because of the large (very large
> > compared to an 897) field of view. Yes, the sCMOS isn't as sensitive
> > as emCCD in the super low photon/pixel limit ( at least on paper),
> > which is why I've reached out to get some real world opinions. We
> > currently don't use any binning on the emCCD. I should mention out
> > emCCD's are ~ 10 years old so I"m sure they aren't as sensitive as
> > they used to be. It sounds like we would be ok replacing an emccd with
> > the scmos, but I'd still like to hear from someone who has done single
> > fluorophore tracking with an sCMOS and how it compared to emCCD.
> >
> > - Jeff
> >
>

dib

Re: [EXT] AW: back illuminates sCMOS vs emCCD

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Hi Jeff,

I have ordered an Andor iXon897 just a few days back. I asked the same
question, researched, talked with many experts, talked with vendors from
both Hamamatsu and Andor, tried to understand the latest technology, and
finally decided to buy EMCCD. BTW, I used several EMCCDs extensively in the
last eight years, specifically for single-molecule tracking,
single-molecule FRET, and ion-sensing at diffusing conditions. Here are
some of my personal opinion/thoughts from the perspective of an extensive
user of EMCCD:

(1) EMCCD is still the gold standard for single-molecule tracking and
single-molecule FRET experiments. A high frame rate with high-sensitivity
is essential for a diffusing single-molecule to get a complete track-patch
(dynamics) with a small step size (time/frame). Here, sensitivity is
critical, and sCMOS does not simply match EMCCD for single-molecule
tracking with fast dynamics. You can go to a high frame rate only if camera
sensitivity is increased. You may get tracking data with sCMOS, but I would
assume it will do at a much lower frame rate to reproduce similar quality
data where dynamics data will be compromised. Or, you have to use a
high-power laser to get fast frame-rate, which can cause rapid
photo-bleaching.

(2) If protein/DNA conformation dynamics are relatively slow at immobilized
conditions, then sCMOS and EMCCD will produce similar results because of
recording data with a slow frame rate (long time/frame).

(3) There are few excellent recent publications to study single-molecule
FRET using sCMOS, where the proteins are fixed on glass or cell/virus
membrane and dynamics is slow. They take advantage of a large sensor size,
which can generate tons of data/time-trajectories in a single FRET
experiment. Many groups even use automated algorithms to generate time
trajectories of donors and acceptors. When a molecule is fixed and the
dynamics are slow, sCMOS is fine.

(4) Ca2+ ion-sensing data is very hard to get with a sCMOS, at least in our
case in T-cell. So, we got an Andor iXon 888 and happy with it.

(5) Do not focus on QE because they are the same (~95%) in both cases.
Better, I would focus on my critical applications to decide EMCCD vs sCMOS-
fast vs slow dynamics or conformational change.

Overall, EMCCD is still the best for all kinds of single-molecule
experiments including diffusing single-molecules, in my personal opinion.

Good luck!

Dibyendu

----
Dibyendu K. Sasmal, PhD
Assistant Professor
Department of Chemistry
Indian Institute of Technology Jodhpur
Jodhpur, RJ 342037, India

M: (+91)-9928-137-008
E: [hidden email]
W: www.sasmallab.org

On Wed, Feb 17, 2021 at 11:10 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 Gerhard,
>
> Gain doesn't reduce the quantum efficiency of the pixel, rather it reduces
> the effective quantum efficiency from the point of view of the user, that
> is the QE you would calculate if you measured your shot noise for a known
> number of photons. For example, suppose your sensor has a QE of 50% and an
> excess noise factor of 1. If you illuminate it with 10 photons per pixel,
> you would measure a shot noise limited snr of sqrt(5) and conclude that the
> QE is 50%. Similarly for a detector with a 100% nominal QE with an ENF of
> 2, 10 photons would also give you a sqrt(5), leading you to again measure a
> 50% QE. This measurement is wrong from the point of view of the pixel (it
> detects every photon), but from a user perspective looking at the whole
> system, the sensor functions as if the QE is 50%. An alternative way to
> think about it is that while the pixel is converting every photon into a
> photoelectron, the stochastic nature of the electron multiplication process
> results in some of those photoelectrons failing to contribute to your SNR.
>
> This is similar to the situation with PMTs, where the QE of the
> photocathode is specified in the datasheet, but if you measure shot noise,
> you'll typically calculate that the QE is about half the value in the
> datasheet due to various losses and noise sources in the electron
> multiplication process (after the photocathode).
>
> Mike
>
> On Wed, Feb 17, 2021 at 11:52 AM Gerhard Holst <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=WAvC5yKfqp8z6AtH8KOwB2qkOooJ8eF7PZdBFQSJbf0&s=qf-L1Gc3xO1zA2ZFq_31JjzoUS6tSbUff_u7-adCl3Y&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=WAvC5yKfqp8z6AtH8KOwB2qkOooJ8eF7PZdBFQSJbf0&s=6rlHnYMhjGUPYb6Xu1yeOPUi70l-V7rsYydsfnlXkxk&e=
> > and include the link in your posting.
> > *****
> >
> > Hi Mike,
> >
> > As far as I know the QE curves in emCCDs are specified with gain = 1,
> > because usually the quantum efficiency refers to the pixels which detect
> > the light and not the gain registers. I absolutely agree, that the
> > additional noise factors have to be taken into account, but the gain
> > doesn't reduce the quantum efficiency, but it reduces the usable
> > intra-scene dynamic range. The higher readout noise of the emCCDs is more
> > than compensated by the gain before readout.
> >
> > In my understanding, the quantum efficiency of such a detector (assuming
> a
> > linear detector model, and assuming that we are above 250nm, such that
> one
> > photon can generate one charge carrier at max) how many charge carriers,
> > let's say electrons, I get from so many photons hitting the detector.
> >
> > Best,
> > Gerhard
> >
> >
> > Dr. Gerhard Holst
> > Head of Research & Science
> > Business Development
> > +49 (0) 9441 2005 570
> >
> > PCO AG, Donaupark 11, 93309 Kelheim, Germany,
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.pco.de&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=WAvC5yKfqp8z6AtH8KOwB2qkOooJ8eF7PZdBFQSJbf0&s=lQdVIGx-B_6lGDhsrzDduA5xZHLQcpopKZfSCji6__8&e=
> > Vorstand / Managing Board: Alexander Grünig, Luitpold Kaspar
> > Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Dr.
> > Emil Ott
> > USt. ID-Nr. / VAT: DE128590843, Registergericht / Register Court:
> > Amtsgericht Regensburg HRB 9157
> > Sitz der Gesellschaft / Registered Office: Kelheim
> >
> >
> >
> > -----Ursprüngliche Nachricht-----
> > Von: Confocal Microscopy List <[hidden email]> Im
> > Auftrag von Michael Giacomelli
> > Gesendet: Mittwoch, 17. Februar 2021 16:48
> > An: [hidden email]
> > Betreff: Re: [EXT] AW: back illuminates sCMOS vs emCCD
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=WAvC5yKfqp8z6AtH8KOwB2qkOooJ8eF7PZdBFQSJbf0&s=qf-L1Gc3xO1zA2ZFq_31JjzoUS6tSbUff_u7-adCl3Y&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=WAvC5yKfqp8z6AtH8KOwB2qkOooJ8eF7PZdBFQSJbf0&s=6rlHnYMhjGUPYb6Xu1yeOPUi70l-V7rsYydsfnlXkxk&e=
> > and include the link in your posting.
> > *****
> >
> > Hi Gerhard,
> >
> > Care is needed when comparing QE figures given the differences between
> how
> > these sensors operate. An EMCCD has internal gain, which means it has
> > excess noise (similar to a PMT) . The effective QE (that is, the QE you
> > would calculate if you measured your shot noise for a known number of
> > photons) is lower than the nominal QE because the gain process introduces
> > additional shot noise. I have no personal experience with these sensors,
> > but Hamamatsu specs an excess noise factor of (1.4)^2 for their EMCCDs.
> > Thus the effective QE would be divided by that factor, giving ~95% with
> EM
> > off, but only ~ 50% with EM on.
> >
> > I saw Fairchild has announced new sCMOS sensors that obtain sub-electron
> > read noise. It does not seem like they're shipping these yet, but I
> think
> > eventually these will replace EMCCD since they should have similar low
> > light sensitivity to EMCCD without the excess noise.
> >
> > Mike
> >
> > On Wed, Feb 17, 2021 at 2:35 AM Gerhard Holst <[hidden email]>
> > wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=qG
> > > msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> > > JVnnxrlDLNPkGMfko&e=
> > > Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > > IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPa
> > > EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> > > and include the link in your posting.
> > > *****
> > >
> > > Hi Jeff,
> > >
> > > the emCCDs with their inbuild gain are nice and also many optical
> > > systems for microscopes were optimized for the large pixel sizes. You
> > > use sensitive as term to describe how good your emCCDs react to light.
> > > In case they are old, and continuously used, I would be more worried
> > > about their gain, because the nice effect of impact ionization is as
> > > well continuously destroying the semiconductor lattice, that's way the
> > > gain curves in emCCD cameras are changing with aging.
> > >
> > > Maybe, for your existing typical application you could do some rough
> > > back-calculations to estimate how many photons you usually "see". As
> > > rule of thumb I would say if you signal per pixel is more than 10
> > > photons, then sCMOS can be a replacement. Below that still the gain of
> > > the emCCDs has an advantage.
> > >
> > > In terms of quantum efficiency (if you call that the "sensitivity") of
> > > the image sensor, there is no difference, maybe the spectral position
> > > of the maximum might be slightly different, but for both types of
> > > image sensors you will find versions with 80 - 95% QE (which are best
> > > possible measured with +/- 5%), so the sensitivity of the image
> > > detector is not the major issue.
> > >
> > > What can be more critical is your optical situation, which corresponds
> > > to what RJ has written. If you want a plugin replacement for your
> > > emCCD camera, you would need a camera with the same pixel size. If you
> > > replace the existing camera with for example 11um pixel size by a
> > > camera with 6.5 um pixel size you will roughly get a 1/4 of the signal
> > > just by geometry and not sensitivity. If you want to use such a
> > > camera, you will need to reduce your illumination circle to increase
> > > the "photon density". Then you could exploit the lower readout noise
> and
> > higher speed of the sCMOS cameras.
> > >
> > > I am not going to repeat all the comparisons of advantages /
> > > disadvantages between emCCD and sCMOS, there is a lot of well written
> > > literature out there, at the end you need the best possible camera for
> > your measurements.
> > >
> > > Regards,
> > > Gerhard
> > >
> > >
> > >
> > >
> > > Dr. Gerhard Holst
> > > Head of Research & Science
> > > Business Development
> > > +49 (0) 9441 2005 570
> > >
> > > PCO AG, Donaupark 11, 93309 Kelheim, Germany,
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.pco.de&d=DwIGa
> > > Q&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOI
> > > XyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&
> > > s=8ySLjREt6J_XNvS7vfaDK_Jt3OJT8YGQHmE8-cDsdg0&e=
> > > Vorstand / Managing Board: Alexander Grünig, Luitpold Kaspar
> > > Vorsitzender des Aufsichtsrats / Chairman of the Supervisory Board: Dr.
> > > Emil Ott
> > > USt. ID-Nr. / VAT: DE128590843, Registergericht / Register Court:
> > > Amtsgericht Regensburg HRB 9157
> > > Sitz der Gesellschaft / Registered Office: Kelheim
> > >
> > >
> > >
> > > -----Ursprüngliche Nachricht-----
> > > Von: Confocal Microscopy List <[hidden email]> Im
> > > Auftrag von Jeff Spector
> > > Gesendet: Dienstag, 16. Februar 2021 19:38
> > > An: [hidden email]
> > > Betreff: Re: back illuminates sCMOS vs emCCD
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=qG
> > > msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> > > JVnnxrlDLNPkGMfko&e=
> > > Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > > IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPa
> > > EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> > > and include the link in your posting.
> > > *****
> > >
> > > Hi,
> > > Thanks for all the advice so far. Please keep it coming. To answer a
> > > few questions :
> > >
> > > On Tue, Feb 16, 2021 at 10:43 AM RJ <[hidden email]> wrote:
> > >
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > >
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=qG
> > > msY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPaEE&s=nrf__InXcGF7l7xeH8KB_FDIOc
> > > JVnnxrlDLNPkGMfko&e=
> > > > Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > > IGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=qGmsY9SfPeSAKcw81X6RuTATT867d03yX9tpmdBPa
> > > EE&s=x2zVHnyBVq2270mPuMhD1kPZlcod70_hKZlPqOmtsmg&e=
> > > and include the link in your posting.
> > > > *****
> > > >
> > > > There is nothing quite like the EMCCD 897 so I think you will be
> > > > disappointed to find an alternative.
> > > > What is your motivation? More gain? Not going to find it.
> > > >
> > > > More resolution or higher speed? These are possible.
> > > >
> > > > Look beyond the max QE. The response curves are different and you
> > > > will also miss the EM gain. The enormous pixels of the 897 hurt the
> > > > spatial resolution but help its sensitivity, take caution if you
> > > > have to software bin to get things where you need on cameras with
> > smaller pixels.
> > > >
> > >
> > > We were drawn to the sCMOS mainly because of the large (very large
> > > compared to an 897) field of view. Yes, the sCMOS isn't as sensitive
> > > as emCCD in the super low photon/pixel limit ( at least on paper),
> > > which is why I've reached out to get some real world opinions. We
> > > currently don't use any binning on the emCCD. I should mention out
> > > emCCD's are ~ 10 years old so I"m sure they aren't as sensitive as
> > > they used to be. It sounds like we would be ok replacing an emccd with
> > > the scmos, but I'd still like to hear from someone who has done single
> > > fluorophore tracking with an sCMOS and how it compared to emCCD.
> > >
> > > - Jeff
> > >
> >
>