localization precision in PALM/STORM

[lechristophe]

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Hi,

Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.

I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.

I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.

So my question is :  How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.

Thanks for your help,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France



 

 

[Mark Cannell]

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Doesn't the quantEM have a photon calibration function? The background noise should be estimated from the variance of the background (extracted from image regions when/where flashes were not detected...). You can also calibrate the camera with weak sources to double check the manufactures stated read-out calibration.

Hope this helps

Mark


On 24/01/2012, at 9:26 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>
> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.
>
> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>
> So my question is :  How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>
> Thanks for your help,
>
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>
>
>
>
>
>

[Guy Cox-2]

Generally one records PALM / STORM images in photon counting mode.  That means that signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded.  So there should be no noise level to worry about and each individual image should be classed by your software as a 0 (no photon) or a 1 (a photon).  The number of counts for that one photon, in a single frame, are meaningless.  To estimate the photon counts for a point in the image you need to see in how many frames that point is scored as a 1.  I hope this makes sense!

                                 Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Tuesday, 24 January 2012 8:27 PM
To: [hidden email]
Subject: localization precision in PALM/STORM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi,

Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.

I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.

I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.

So my question is :  How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.

Thanks for your help,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France



 

 

[lechristophe]

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Hi Guy,  

Not sure I understand what you mean here, I don't think I'm using "photon count" in my experiment. It's a wide field setup and I'm acquiring streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz framerate, with a magnified pixel size around 100 nm. The conditions are optimized for acquiring a whole photon burst (500-3000 photons depending on the fluorophore) in one, max two images, spread on a few pixels, to have a good signal.

Do I have to ask Photometrics for a calibration curve between intensity levels and photoelectrons (and relate to photons using the quantum efficiency curve) ? I couldn't find that info on their website.

Thanks for your help,

Christophe


Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :  

> Generally one records PALM / STORM images in photon counting mode. That means that signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded. So there should be no noise level to worry about and each individual image should be classed by your software as a 0 (no photon) or a 1 (a photon). The number of counts for that one photon, in a single frame, are meaningless. To estimate the photon counts for a point in the image you need to see in how many frames that point is scored as a 1. I hope this makes sense!
>  
> Guy
>  
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate,  
> Australian Centre for Microscopy & Microanalysis,  
> Madsen Building F09, University of Sydney, NSW 2006  
>  
> Phone [phone] Fax [phone]
> Mobile [phone]
> ______________________________________________
> http://www.guycox.net
>  
>  
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
> Sent: Tuesday, 24 January 2012 8:27 PM
> To: [hidden email] (mailto:[hidden email])
> Subject: localization precision in PALM/STORM
>  
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>  
> Hi,  
>  
> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>  
> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
>  
> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>  
> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>  
> Thanks for your help,
>  
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>  
>  
>  
>  

[lechristophe]

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*****

Hi Mark,  

I don't think the quantEM has a built-in photon calibration function, in contrast to the newer Evolve camera, also from Photometrics. The spec sheet for the quantEM is available here :
http://www.photometrics.com/products/datasheets/qem512sc.pdf

Do I have to calibrate it myself or is Photometrics supposed to provide a photon/intensity calibration curve? I don't want exact experimental values for my precise camera, just a reasonable estimate to derive a theoretical "best" value for localization accuracy.

Christophe  


Le mardi 24 janvier 2012 à 10:58, Mark Cannell a écrit :

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>  
> Doesn't the quantEM have a photon calibration function? The background noise should be estimated from the variance of the background (extracted from image regions when/where flashes were not detected...). You can also calibrate the camera with weak sources to double check the manufactures stated read-out calibration.
>  
> Hope this helps
>  
> Mark
>  
>  
> On 24/01/2012, at 9:26 AM, Christophe Leterrier wrote:
>  
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >  
> > Hi,  
> >  
> > Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
> >  
> > I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
> >  
> > I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
> >  
> > So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
> >  
> > Thanks for your help,
> >  
> > --
> > Christophe Leterrier
> > Researcher
> > Axonal Domains Architecture Team
> > CRN2M CNRS UMR 7286
> > Aix Marseille University, France
> >  
>  
>  
>  

[Roger Phillips]

The new scientific CMOS cameras are touted as applicable to single molecule localization microscopy.  But I find no mention of a photon counting mode.  How are photon counts made with these cameras?
Roger

Dr Roger Guy Phillips
Centre for Advanced Microscopy,
University of Sussex
School of Life Sciences
John Maynard Smith Building
Falmer, Brighton & Hove
BN1 9QG
United Kingdom
 
phone:[phone]
fax: [phone]
email: [hidden email]
room:2C9 (ext 7585)/lab 4C2 (ext 2734) 




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: 24 January 2012 13:04
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Guy,  

Not sure I understand what you mean here, I don't think I'm using "photon count" in my experiment. It's a wide field setup and I'm acquiring streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz framerate, with a magnified pixel size around 100 nm. The conditions are optimized for acquiring a whole photon burst (500-3000 photons depending on the fluorophore) in one, max two images, spread on a few pixels, to have a good signal.

Do I have to ask Photometrics for a calibration curve between intensity levels and photoelectrons (and relate to photons using the quantum efficiency curve) ? I couldn't find that info on their website.

Thanks for your help,

Christophe


Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :  

> Generally one records PALM / STORM images in photon counting mode. That means that signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded. So there should be no noise level to worry about and each individual image should be classed by your software as a 0 (no photon) or a 1 (a photon). The number of counts for that one photon, in a single frame, are meaningless. To estimate the photon counts for a point in the image you need to see in how many frames that point is scored as a 1. I hope this makes sense!
>  
> Guy
>  
> Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press /
> Taylor & Francis http://www.guycox.com/optical.htm 
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
> NSW 2006
>  
> Phone [phone] Fax [phone] Mobile [phone]
> ______________________________________________
> http://www.guycox.net
>  
>  
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Christophe
> Leterrier
> Sent: Tuesday, 24 January 2012 8:27 PM
> To: [hidden email]
> (mailto:[hidden email])
> Subject: localization precision in PALM/STORM
>  
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>  
> Hi,
>  
> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>  
> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
>  
> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>  
> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>  
> Thanks for your help,
>  
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>  
>  
>  
>  

[Christian Soeller]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

You basically want to convert the AD counts in your image into photon numbers. That requires several bits of info about the camera. We have Andor cameras and I can look up the required values from the data/test sheet that comes with the individual camera (these change from cam to cam). You need to know the
- electrons per count
- absolute EM gain (with the quantEM you might have to measure/calibrate this)
- A/D offset (this can be measured in dark frames with no light impinging on the cam, i.e. shutter closed)

The formula is then something like

photons = (counts - A/D offset)*electrons-per-count/EM-gain

Some more recent camera software packages may have functions to make this conversion for you. I am not sure about photometrics cams/SDKs.

Due the additional noise introduced by the EM gain process you should divide the resulting photon-numbers by 2 before looking up values from the Thompson et al. formula. There are papers on EM CCDs that explain this. I also seem to recall that some more recent papers have a subtle correction to the Thompson formula.

Hope this helps,

Christian

On 25/01/2012, at 2:08 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Mark,  
>
> I don't think the quantEM has a built-in photon calibration function, in contrast to the newer Evolve camera, also from Photometrics. The spec sheet for the quantEM is available here :
> http://www.photometrics.com/products/datasheets/qem512sc.pdf
>
> Do I have to calibrate it myself or is Photometrics supposed to provide a photon/intensity calibration curve? I don't want exact experimental values for my precise camera, just a reasonable estimate to derive a theoretical "best" value for localization accuracy.
>
> Christophe  
>
>
> Le mardi 24 janvier 2012 à 10:58, Mark Cannell a écrit :
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Doesn't the quantEM have a photon calibration function? The background noise should be estimated from the variance of the background (extracted from image regions when/where flashes were not detected...). You can also calibrate the camera with weak sources to double check the manufactures stated read-out calibration.
>>
>> Hope this helps
>>
>> Mark
>>
>>
>> On 24/01/2012, at 9:26 AM, Christophe Leterrier wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi,  
>>>
>>> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>>>
>>> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
>>>
>>> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>>>
>>> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>>>
>>> Thanks for your help,
>>>
>>> --
>>> Christophe Leterrier
>>> Researcher
>>> Axonal Domains Architecture Team
>>> CRN2M CNRS UMR 7286
>>> Aix Marseille University, France
>>>
>>
>>
>>

--
Christian Soeller PhD   Dept. of Physiology  [phone] x82770
University of Auckland  Auckland, New Zealand  fax [phone]

[Christian Soeller]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

All cameras effectively run in a photon counting mode (more precisely they count the photo-electrons). The sCMOS cams basically have no EM gain but low enough read-out noise that it is not strictly necessary with typical photon counts. We have briefly tested a Andor Neo and it seems to work.

Christian

On 25/01/2012, at 3:29 AM, Roger Phillips wrote:

> The new scientific CMOS cameras are touted as applicable to single molecule localization microscopy.  But I find no mention of a photon counting mode.  How are photon counts made with these cameras?
> Roger
>
> Dr Roger Guy Phillips
> Centre for Advanced Microscopy,
> University of Sussex
> School of Life Sciences
> John Maynard Smith Building
> Falmer, Brighton & Hove
> BN1 9QG
> United Kingdom
>  
> phone:[phone]
> fax: [phone]
> email: [hidden email]
> room:2C9 (ext 7585)/lab 4C2 (ext 2734)  
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
> Sent: 24 January 2012 13:04
> To: [hidden email]
> Subject: Re: localization precision in PALM/STORM
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Guy,  
>
> Not sure I understand what you mean here, I don't think I'm using "photon count" in my experiment. It's a wide field setup and I'm acquiring streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz framerate, with a magnified pixel size around 100 nm. The conditions are optimized for acquiring a whole photon burst (500-3000 photons depending on the fluorophore) in one, max two images, spread on a few pixels, to have a good signal.
>
> Do I have to ask Photometrics for a calibration curve between intensity levels and photoelectrons (and relate to photons using the quantum efficiency curve) ? I couldn't find that info on their website.
>
> Thanks for your help,
>
> Christophe
>
>
> Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :  
>> Generally one records PALM / STORM images in photon counting mode. That means that signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded. So there should be no noise level to worry about and each individual image should be classed by your software as a 0 (no photon) or a 1 (a photon). The number of counts for that one photon, in a single frame, are meaningless. To estimate the photon counts for a point in the image you need to see in how many frames that point is scored as a 1. I hope this makes sense!
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press /
>> Taylor & Francis http://www.guycox.com/optical.htm 
>> ______________________________________________
>> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
>> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
>> NSW 2006
>>
>> Phone [phone] Fax [phone] Mobile [phone]
>> ______________________________________________
>> http://www.guycox.net
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Christophe
>> Leterrier
>> Sent: Tuesday, 24 January 2012 8:27 PM
>> To: [hidden email]
>> (mailto:[hidden email])
>> Subject: localization precision in PALM/STORM
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi,
>>
>> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>>
>> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
>>
>> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>>
>> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>>
>> Thanks for your help,
>>
>> --
>> Christophe Leterrier
>> Researcher
>> Axonal Domains Architecture Team
>> CRN2M CNRS UMR 7286
>> Aix Marseille University, France
>>
>>
>>
>>

--
Christian Soeller PhD   Dept. of Physiology  [phone] x82770
University of Auckland  Auckland, New Zealand  fax [phone]

[Roger Phillips]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks, Christian,
So the sCMOS photon count would come from the formula
photons = (counts - A/D offset)*electrons-per-count/EM-gain
with EM-gain set to 1 and no 'additional noise introduced by the EM gain process'?

In Guy's reply, he said 'signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded.'  This methods seems to account for the 'low threshold' but not for the possible 'pile-up'?
Roger

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Soeller
Sent: 24 January 2012 14:44
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

You basically want to convert the AD counts in your image into photon numbers. That requires several bits of info about the camera. We have Andor cameras and I can look up the required values from the data/test sheet that comes with the individual camera (these change from cam to cam). You need to know the
- electrons per count
- absolute EM gain (with the quantEM you might have to measure/calibrate this)
- A/D offset (this can be measured in dark frames with no light impinging on the cam, i.e. shutter closed)

The formula is then something like

photons = (counts - A/D offset)*electrons-per-count/EM-gain

Some more recent camera software packages may have functions to make this conversion for you. I am not sure about photometrics cams/SDKs.

Due the additional noise introduced by the EM gain process you should divide the resulting photon-numbers by 2 before looking up values from the Thompson et al. formula. There are papers on EM CCDs that explain this. I also seem to recall that some more recent papers have a subtle correction to the Thompson formula.

Hope this helps,

Christian

On 25/01/2012, at 2:08 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Mark,  
>
> I don't think the quantEM has a built-in photon calibration function, in contrast to the newer Evolve camera, also from Photometrics. The spec sheet for the quantEM is available here :
> http://www.photometrics.com/products/datasheets/qem512sc.pdf
>
> Do I have to calibrate it myself or is Photometrics supposed to provide a photon/intensity calibration curve? I don't want exact experimental values for my precise camera, just a reasonable estimate to derive a theoretical "best" value for localization accuracy.
>
> Christophe  
>
>
> Le mardi 24 janvier 2012 à 10:58, Mark Cannell a écrit :
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Doesn't the quantEM have a photon calibration function? The background noise should be estimated from the variance of the background (extracted from image regions when/where flashes were not detected...). You can also calibrate the camera with weak sources to double check the manufactures stated read-out calibration.
>>
>> Hope this helps
>>
>> Mark
>>
>>
>> On 24/01/2012, at 9:26 AM, Christophe Leterrier wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi,  
>>>
>>> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>>>
>>> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
>>>
>>> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>>>
>>> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>>>
>>> Thanks for your help,
>>>
>>> --
>>> Christophe Leterrier
>>> Researcher
>>> Axonal Domains Architecture Team
>>> CRN2M CNRS UMR 7286
>>> Aix Marseille University, France
>>>
>>
>>
>>

--
Christian Soeller PhD   Dept. of Physiology  [phone] x82770
University of Auckland  Auckland, New Zealand  fax [phone]

[Christian Soeller]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Essentially yes.

Guy's reply would seem to apply to photon-counting with PMTs when run in photon-counting-mode, not to cameras. Guy, please correct me if I am wrong.

Christian

On 25/01/2012, at 4:18 AM, Roger Phillips wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks, Christian,
> So the sCMOS photon count would come from the formula
> photons = (counts - A/D offset)*electrons-per-count/EM-gain
> with EM-gain set to 1 and no 'additional noise introduced by the EM gain process'?
>
> In Guy's reply, he said 'signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded.'  This methods seems to account for the 'low threshold' but not for the possible 'pile-up'?
> Roger
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Soeller
> Sent: 24 January 2012 14:44
> To: [hidden email]
> Subject: Re: localization precision in PALM/STORM
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> You basically want to convert the AD counts in your image into photon numbers. That requires several bits of info about the camera. We have Andor cameras and I can look up the required values from the data/test sheet that comes with the individual camera (these change from cam to cam). You need to know the
> - electrons per count
> - absolute EM gain (with the quantEM you might have to measure/calibrate this)
> - A/D offset (this can be measured in dark frames with no light impinging on the cam, i.e. shutter closed)
>
> The formula is then something like
>
> photons = (counts - A/D offset)*electrons-per-count/EM-gain
>
> Some more recent camera software packages may have functions to make this conversion for you. I am not sure about photometrics cams/SDKs.
>
> Due the additional noise introduced by the EM gain process you should divide the resulting photon-numbers by 2 before looking up values from the Thompson et al. formula. There are papers on EM CCDs that explain this. I also seem to recall that some more recent papers have a subtle correction to the Thompson formula.
>
> Hope this helps,
>
> Christian
>
> On 25/01/2012, at 2:08 AM, Christophe Leterrier wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Mark,  
>>
>> I don't think the quantEM has a built-in photon calibration function, in contrast to the newer Evolve camera, also from Photometrics. The spec sheet for the quantEM is available here :
>> http://www.photometrics.com/products/datasheets/qem512sc.pdf
>>
>> Do I have to calibrate it myself or is Photometrics supposed to provide a photon/intensity calibration curve? I don't want exact experimental values for my precise camera, just a reasonable estimate to derive a theoretical "best" value for localization accuracy.
>>
>> Christophe  
>>
>>
>> Le mardi 24 janvier 2012 à 10:58, Mark Cannell a écrit :
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Doesn't the quantEM have a photon calibration function? The background noise should be estimated from the variance of the background (extracted from image regions when/where flashes were not detected...). You can also calibrate the camera with weak sources to double check the manufactures stated read-out calibration.
>>>
>>> Hope this helps
>>>
>>> Mark
>>>
>>>
>>> On 24/01/2012, at 9:26 AM, Christophe Leterrier wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi,  
>>>>
>>>> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>>>>
>>>> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
>>>>
>>>> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>>>>
>>>> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>>>>
>>>> Thanks for your help,
>>>>
>>>> --
>>>> Christophe Leterrier
>>>> Researcher
>>>> Axonal Domains Architecture Team
>>>> CRN2M CNRS UMR 7286
>>>> Aix Marseille University, France
>>>>
>>>
>>>
>>>
>

[Andrew York]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Expose your camera to different constant, uniform light levels, perhaps
with a brightfield lamp. Take a bunch of pictures at each light level. For
a given pixel, plot variance in signal vs. mean signal at each light level.
The shape of this curve should tell you how many A/D counts you get per
photoelectron, since photoelectron counting is hopefully a Poisson process.
Bonus points: Do all your pixels agree on the relationship between variance
and mean?

Devil's advocate: suppose you do your calibration wrong. How would you ever
tell? There ought to be a good answer for this. If there's no way you could
ever tell you were wrong, what are you accomplishing?

On Tue, Jan 24, 2012 at 9:51 AM, Christian Soeller <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> All cameras effectively run in a photon counting mode (more precisely they
> count the photo-electrons). The sCMOS cams basically have no EM gain but
> low enough read-out noise that it is not strictly necessary with typical
> photon counts. We have briefly tested a Andor Neo and it seems to work.
>
> Christian
>
> On 25/01/2012, at 3:29 AM, Roger Phillips wrote:
>
> > The new scientific CMOS cameras are touted as applicable to single
> molecule localization microscopy.  But I find no mention of a photon
> counting mode.  How are photon counts made with these cameras?
> > Roger
> >
> > Dr Roger Guy Phillips
> > Centre for Advanced Microscopy,
> > University of Sussex
> > School of Life Sciences
> > John Maynard Smith Building
> > Falmer, Brighton & Hove
> > BN1 9QG
> > United Kingdom
> >
> > phone:[phone]
> > fax: [phone]
> > email: [hidden email]
> > room:2C9 (ext 7585)/lab 4C2 (ext 2734)
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Christophe Leterrier
> > Sent: 24 January 2012 13:04
> > To: [hidden email]
> > Subject: Re: localization precision in PALM/STORM
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Guy,
> >
> > Not sure I understand what you mean here, I don't think I'm using
> "photon count" in my experiment. It's a wide field setup and I'm acquiring
> streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz
> framerate, with a magnified pixel size around 100 nm. The conditions are
> optimized for acquiring a whole photon burst (500-3000 photons depending on
> the fluorophore) in one, max two images, spread on a few pixels, to have a
> good signal.
> >
> > Do I have to ask Photometrics for a calibration curve between intensity
> levels and photoelectrons (and relate to photons using the quantum
> efficiency curve) ? I couldn't find that info on their website.
> >
> > Thanks for your help,
> >
> > Christophe
> >
> >
> > Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :
> >> Generally one records PALM / STORM images in photon counting mode. That
> means that signals below a certain threshold are regarded as noise, and
> discarded, and signals above a higher threshold are regarded as 'pile-up'
> and also discarded. So there should be no noise level to worry about and
> each individual image should be classed by your software as a 0 (no photon)
> or a 1 (a photon). The number of counts for that one photon, in a single
> frame, are meaningless. To estimate the photon counts for a point in the
> image you need to see in how many frames that point is scored as a 1. I
> hope this makes sense!
> >>
> >> Guy
> >>
> >> Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press /
> >> Taylor & Francis http://www.guycox.com/optical.htm
> >> ______________________________________________
> >> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
> >> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
> >> NSW 2006
> >>
> >> Phone [phone] Fax [phone] Mobile [phone]
> >> ______________________________________________
> >> http://www.guycox.net
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >> [mailto:[hidden email]] On Behalf Of Christophe
> >> Leterrier
> >> Sent: Tuesday, 24 January 2012 8:27 PM
> >> To: [hidden email]
> >> (mailto:[hidden email])
> >> Subject: localization precision in PALM/STORM
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hi,
> >>
> >> Not strictly a confocal question, but I'm pretty sure this list is the
> best place to get thorough and insightful answers.
> >>
> >> I have made 2D STORM (stochastic optical reconstruction microscopy)
> acquisitions and processing and I end up with a table of XY localized
> fluorophores together with the integrated intensity of the localized
> diffraction-limited spot.
> >>
> >> I'd like to plot each fluorophore as a gaussian with a width
> corresponding to the localization precision, similar to what was done in
> Bates et al. Science 2007. According to equation (17) in Thompson, Larson &
> Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the
> number of photons collected, the width of the diffraction-limited spot, the
> size of the camera pixel, and the background noise.
> >>
> >> So my question is : How do I get the number of photons from the
> intensity level of an image? I'm using a Photometrics 512*512 QuantEM
> camera. What is the background noise and how do I estimate it? Then using
> these values in the Thompson et al. equation, I can get a theoretical spot
> intensity / localization precision calibration curve that I could use for
> the gaussian-based reconstruction.
> >>
> >> Thanks for your help,
> >>
> >> --
> >> Christophe Leterrier
> >> Researcher
> >> Axonal Domains Architecture Team
> >> CRN2M CNRS UMR 7286
> >> Aix Marseille University, France
> >>
> >>
> >>
> >>
>
> --
> Christian Soeller PhD   Dept. of Physiology  [phone] x82770
> University of Auckland  Auckland, New Zealand  fax [phone]
>

[Nico Stuurman]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Christoph,

> I don't think the quantEM has a built-in photon calibration function, in contrast to the newer Evolve camera, also from Photometrics. The spec sheet for the quantEM is available here :
> http://www.photometrics.com/products/datasheets/qem512sc.pdf
>
> Do I have to calibrate it myself or is Photometrics supposed to provide a photon/intensity calibration curve?

It is certainly worth your effort to determine the photo-electron-conversion factor yourself.  It is actually quite simple to do, and it is always good to validate the equipment your work with.

I wrote a detailed protocol and software scripts for Micro-Manager that guide you through the measurements:

http://valelab.ucsf.edu/~MM/MMwiki/index.php/Measuring_camera_specifications



Hope this helps!


Nico

[Jean-Yves Tinevez-3]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

On 24-Jan-12 15:43, Christian Soeller wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> You basically want to convert the AD counts in your image into photon numbers. That requires several bits of info about the camera. We have Andor cameras and I can look up the required values from the data/test sheet that comes with the individual camera (these change from cam to cam). You need to know the
> - electrons per count
> - absolute EM gain (with the quantEM you might have to measure/calibrate this)
> - A/D offset (this can be measured in dark frames with no light impinging on the cam, i.e. shutter closed)
>

Dear all,

Christophe wants to estimate the localization precision dependance on
the detected photons, so only the photo-electrons matter. Therefore, you
can simply deduce what they are for each pixel using simple pixel
fluctuation, or variance estimates. You do not need the quantum efficiency.

Best
jyt


--
Jean-Yves Tinevez
PFID - Imagopole
Institut Pasteur
25-28, rue du Docteur Roux
75724 Paris cedex 15
France
tel: [phone]

[Hendrik Deschout]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello Christophe,

If your camera does not show photon counts, you can easily determine the conversion factor between the pixel intensity and the number of photons yourself, as mentioned by Andrew. The approach exploits the Poisson statistics of the photon numbers, see also "Scientific Charge-Coupled Devices" by Janesick.

The interpretation of the background term "b" in the Thompson formula is somewhat tricky. Someone once pointed out to me that, in case you approximate the PSF by a two-dimensional Gaussian, it is the number of photons that comes from the background, plus the shoulders of the PSF which are not described by the Gaussian. If you again assume a Poisson distribution for the photon numbers, "b^2" can be determined as the variance of the background pixel intensities near the PSF (corrected with the conversion factor of course).

By the way, I would like to note that the Thompson formula (and in particular the 30% error) has been corrected by Mortensen et al. in their very nice Nature Methods paper (2010, vol. 7, no. 5). Also note that the Thompson formula was derived for a CCD camera. In case of an electron multiplying CCD camera, the entire expression for "sigma^2" should be multiplied by 2, the electron multiplication is to blame, this is often ignored. In this case, it also means that the background variance you measure is actually equal to "2*b^2".

With kind regards,

Hendrik

Hendrik Deschout
Biophotonic Imaging Group
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Gent
Belgium
Tel: [phone]
Fax: [phone]
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew York
Sent: dinsdag 24 januari 2012 16:39
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Expose your camera to different constant, uniform light levels, perhaps
with a brightfield lamp. Take a bunch of pictures at each light level. For
a given pixel, plot variance in signal vs. mean signal at each light level.
The shape of this curve should tell you how many A/D counts you get per
photoelectron, since photoelectron counting is hopefully a Poisson process.
Bonus points: Do all your pixels agree on the relationship between variance
and mean?

Devil's advocate: suppose you do your calibration wrong. How would you ever
tell? There ought to be a good answer for this. If there's no way you could
ever tell you were wrong, what are you accomplishing?

On Tue, Jan 24, 2012 at 9:51 AM, Christian Soeller <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> All cameras effectively run in a photon counting mode (more precisely they
> count the photo-electrons). The sCMOS cams basically have no EM gain but
> low enough read-out noise that it is not strictly necessary with typical
> photon counts. We have briefly tested a Andor Neo and it seems to work.
>
> Christian
>
> On 25/01/2012, at 3:29 AM, Roger Phillips wrote:
>
> > The new scientific CMOS cameras are touted as applicable to single
> molecule localization microscopy.  But I find no mention of a photon
> counting mode.  How are photon counts made with these cameras?
> > Roger
> >
> > Dr Roger Guy Phillips
> > Centre for Advanced Microscopy,
> > University of Sussex
> > School of Life Sciences
> > John Maynard Smith Building
> > Falmer, Brighton & Hove
> > BN1 9QG
> > United Kingdom
> >
> > phone:[phone]
> > fax: [phone]
> > email: [hidden email]
> > room:2C9 (ext 7585)/lab 4C2 (ext 2734)
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Christophe Leterrier
> > Sent: 24 January 2012 13:04
> > To: [hidden email]
> > Subject: Re: localization precision in PALM/STORM
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Guy,
> >
> > Not sure I understand what you mean here, I don't think I'm using
> "photon count" in my experiment. It's a wide field setup and I'm acquiring
> streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz
> framerate, with a magnified pixel size around 100 nm. The conditions are
> optimized for acquiring a whole photon burst (500-3000 photons depending on
> the fluorophore) in one, max two images, spread on a few pixels, to have a
> good signal.
> >
> > Do I have to ask Photometrics for a calibration curve between intensity
> levels and photoelectrons (and relate to photons using the quantum
> efficiency curve) ? I couldn't find that info on their website.
> >
> > Thanks for your help,
> >
> > Christophe
> >
> >
> > Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :
> >> Generally one records PALM / STORM images in photon counting mode. That
> means that signals below a certain threshold are regarded as noise, and
> discarded, and signals above a higher threshold are regarded as 'pile-up'
> and also discarded. So there should be no noise level to worry about and
> each individual image should be classed by your software as a 0 (no photon)
> or a 1 (a photon). The number of counts for that one photon, in a single
> frame, are meaningless. To estimate the photon counts for a point in the
> image you need to see in how many frames that point is scored as a 1. I
> hope this makes sense!
> >>
> >> Guy
> >>
> >> Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press /
> >> Taylor & Francis http://www.guycox.com/optical.htm
> >> ______________________________________________
> >> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
> >> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
> >> NSW 2006
> >>
> >> Phone [phone] Fax [phone] Mobile [phone]
> >> ______________________________________________
> >> http://www.guycox.net
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >> [mailto:[hidden email]] On Behalf Of Christophe
> >> Leterrier
> >> Sent: Tuesday, 24 January 2012 8:27 PM
> >> To: [hidden email]
> >> (mailto:[hidden email])
> >> Subject: localization precision in PALM/STORM
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hi,
> >>
> >> Not strictly a confocal question, but I'm pretty sure this list is the
> best place to get thorough and insightful answers.
> >>
> >> I have made 2D STORM (stochastic optical reconstruction microscopy)
> acquisitions and processing and I end up with a table of XY localized
> fluorophores together with the integrated intensity of the localized
> diffraction-limited spot.
> >>
> >> I'd like to plot each fluorophore as a gaussian with a width
> corresponding to the localization precision, similar to what was done in
> Bates et al. Science 2007. According to equation (17) in Thompson, Larson &
> Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the
> number of photons collected, the width of the diffraction-limited spot, the
> size of the camera pixel, and the background noise.
> >>
> >> So my question is : How do I get the number of photons from the
> intensity level of an image? I'm using a Photometrics 512*512 QuantEM
> camera. What is the background noise and how do I estimate it? Then using
> these values in the Thompson et al. equation, I can get a theoretical spot
> intensity / localization precision calibration curve that I could use for
> the gaussian-based reconstruction.
> >>
> >> Thanks for your help,
> >>
> >> --
> >> Christophe Leterrier
> >> Researcher
> >> Axonal Domains Architecture Team
> >> CRN2M CNRS UMR 7286
> >> Aix Marseille University, France
> >>
> >>
> >>
> >>
>
> --
> Christian Soeller PhD   Dept. of Physiology  [phone] x82770
> University of Auckland  Auckland, New Zealand  fax [phone]
>

[Sripad Ram]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello Christophe,
As Hendrik has pointed out, there are issues with using the Thompson formula
and this has been investigated by us and others.

The rigorous approach to calculate the localization accuracy that is based
on the Fisher information matrix was introduced by our group many years ago
(e.g, see Ober et al., Biophys. J. 2004, 86:1185-1200, Ram et al., Proc.
SPIE, 2005, 5699: 426-435, Ram et al., Biophys J. 2008, 95: 6025-6043).

We have a software package to calculate the limit of the localization
accuracy for different image profiles. Please check out our lab webpage for
more info (http://www4.utsouthwestern.edu/wardlab/fandplimittool.asp).

Regards,
Sripad


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Hendrik Deschout
Sent: Tuesday, January 24, 2012 11:08 AM
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello Christophe,

If your camera does not show photon counts, you can easily determine the
conversion factor between the pixel intensity and the number of photons
yourself, as mentioned by Andrew. The approach exploits the Poisson
statistics of the photon numbers, see also "Scientific Charge-Coupled
Devices" by Janesick.

The interpretation of the background term "b" in the Thompson formula is
somewhat tricky. Someone once pointed out to me that, in case you
approximate the PSF by a two-dimensional Gaussian, it is the number of
photons that comes from the background, plus the shoulders of the PSF which
are not described by the Gaussian. If you again assume a Poisson
distribution for the photon numbers, "b^2" can be determined as the variance
of the background pixel intensities near the PSF (corrected with the
conversion factor of course).

By the way, I would like to note that the Thompson formula (and in
particular the 30% error) has been corrected by Mortensen et al. in their
very nice Nature Methods paper (2010, vol. 7, no. 5). Also note that the
Thompson formula was derived for a CCD camera. In case of an electron
multiplying CCD camera, the entire expression for "sigma^2" should be
multiplied by 2, the electron multiplication is to blame, this is often
ignored. In this case, it also means that the background variance you
measure is actually equal to "2*b^2".

With kind regards,

Hendrik

Hendrik Deschout
Biophotonic Imaging Group
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Gent
Belgium
Tel: [phone]
Fax: [phone]
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Andrew York
Sent: dinsdag 24 januari 2012 16:39
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

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Expose your camera to different constant, uniform light levels, perhaps
with a brightfield lamp. Take a bunch of pictures at each light level. For
a given pixel, plot variance in signal vs. mean signal at each light level.
The shape of this curve should tell you how many A/D counts you get per
photoelectron, since photoelectron counting is hopefully a Poisson process.
Bonus points: Do all your pixels agree on the relationship between variance
and mean?

Devil's advocate: suppose you do your calibration wrong. How would you ever
tell? There ought to be a good answer for this. If there's no way you could
ever tell you were wrong, what are you accomplishing?

On Tue, Jan 24, 2012 at 9:51 AM, Christian Soeller <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> All cameras effectively run in a photon counting mode (more precisely they
> count the photo-electrons). The sCMOS cams basically have no EM gain but
> low enough read-out noise that it is not strictly necessary with typical
> photon counts. We have briefly tested a Andor Neo and it seems to work.
>
> Christian
>
> On 25/01/2012, at 3:29 AM, Roger Phillips wrote:
>
> > The new scientific CMOS cameras are touted as applicable to single
> molecule localization microscopy.  But I find no mention of a photon
> counting mode.  How are photon counts made with these cameras?
> > Roger
> >
> > Dr Roger Guy Phillips
> > Centre for Advanced Microscopy,
> > University of Sussex
> > School of Life Sciences
> > John Maynard Smith Building
> > Falmer, Brighton & Hove
> > BN1 9QG
> > United Kingdom
> >
> > phone:[phone]
> > fax: [phone]
> > email: [hidden email]
> > room:2C9 (ext 7585)/lab 4C2 (ext 2734)
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Christophe Leterrier
> > Sent: 24 January 2012 13:04
> > To: [hidden email]
> > Subject: Re: localization precision in PALM/STORM
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Guy,
> >
> > Not sure I understand what you mean here, I don't think I'm using
> "photon count" in my experiment. It's a wide field setup and I'm acquiring
> streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz
> framerate, with a magnified pixel size around 100 nm. The conditions are
> optimized for acquiring a whole photon burst (500-3000 photons depending

on

> the fluorophore) in one, max two images, spread on a few pixels, to have a
> good signal.
> >
> > Do I have to ask Photometrics for a calibration curve between intensity
> levels and photoelectrons (and relate to photons using the quantum
> efficiency curve) ? I couldn't find that info on their website.
> >
> > Thanks for your help,
> >
> > Christophe
> >
> >
> > Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :
> >> Generally one records PALM / STORM images in photon counting mode. That
> means that signals below a certain threshold are regarded as noise, and
> discarded, and signals above a higher threshold are regarded as 'pile-up'
> and also discarded. So there should be no noise level to worry about and
> each individual image should be classed by your software as a 0 (no

photon)

> or a 1 (a photon). The number of counts for that one photon, in a single
> frame, are meaningless. To estimate the photon counts for a point in the
> image you need to see in how many frames that point is scored as a 1. I
> hope this makes sense!
> >>
> >> Guy
> >>
> >> Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press /
> >> Taylor & Francis http://www.guycox.com/optical.htm
> >> ______________________________________________
> >> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
> >> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
> >> NSW 2006
> >>
> >> Phone [phone] Fax [phone] Mobile [phone]
> >> ______________________________________________
> >> http://www.guycox.net
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >> [mailto:[hidden email]] On Behalf Of Christophe
> >> Leterrier
> >> Sent: Tuesday, 24 January 2012 8:27 PM
> >> To: [hidden email]
> >> (mailto:[hidden email])
> >> Subject: localization precision in PALM/STORM
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hi,
> >>
> >> Not strictly a confocal question, but I'm pretty sure this list is the
> best place to get thorough and insightful answers.
> >>
> >> I have made 2D STORM (stochastic optical reconstruction microscopy)
> acquisitions and processing and I end up with a table of XY localized
> fluorophores together with the integrated intensity of the localized
> diffraction-limited spot.
> >>
> >> I'd like to plot each fluorophore as a gaussian with a width
> corresponding to the localization precision, similar to what was done in
> Bates et al. Science 2007. According to equation (17) in Thompson, Larson

&
> Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the
> number of photons collected, the width of the diffraction-limited spot,
the

> size of the camera pixel, and the background noise.
> >>
> >> So my question is : How do I get the number of photons from the
> intensity level of an image? I'm using a Photometrics 512*512 QuantEM
> camera. What is the background noise and how do I estimate it? Then using
> these values in the Thompson et al. equation, I can get a theoretical spot
> intensity / localization precision calibration curve that I could use for
> the gaussian-based reconstruction.
> >>
> >> Thanks for your help,
> >>
> >> --
> >> Christophe Leterrier
> >> Researcher
> >> Axonal Domains Architecture Team
> >> CRN2M CNRS UMR 7286
> >> Aix Marseille University, France
> >>
> >>
> >>
> >>
>
> --
> Christian Soeller PhD   Dept. of Physiology  [phone] x82770
> University of Auckland  Auckland, New Zealand  fax [phone]
>

[James Pawley]

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To join, leave or search the confocal microscopy listserv, go to:
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*****

Hi all,

I have enjoyed the descriptions of how to measure
detector noise contributions in PALM/STORM (and I
am interested to see how the sCMOS cameras
compare with the more common EM-CCD!).

However, while it is certainly important to know
noise levels of the various photodetectors, there
may be other noise sources that may be even more
important.

What about the statistical and other errors
related to the very small numbers of fluorescent
molecules actually localized?

Just because we can localize some fluors, doesn't
mean that those measured are a statistically
random subset of those present. For imaging
setups that use laser light and embedded
specimens, 2/3 of the molecules will be in the
wrong orientation to be significantly excited and
the the chance that the other third will be
excited will vary with their orientation.  These
limits probably apply to both the photoactivation
beam and that used for excitation (Might it be
important to have them both polarized in the same
direction?).

In addition, when TIRF is used, excitation also
varies very strongly with the distance of the
fluor from the glass-water or glass-media
interface and the depth of the evanescent field
varies with pol orientation. In some cases, this
interface itself, may affect fluorophor
orientation. In any case, the chance of exciting
a fluor attached to the near side of a structure
only a few hundred nm in size may be
significantly different from the chance of
exciting a fluorophor on its far side. Won't this
distort the structure's apparent shape?

Even if the fluorescent molecule is not prevented
from rotating by steric constraints, may we
assume that its chance of being excited varies
only with its position in the evanescent field?
What about Brownian and other motions? Won't such
motions blurr the image of moving fluorophors
causing them to be discriminated out by software
looking for nice round Airy spots? (Some software
assumes that ovoid blobs must represent two
nearby fluorophors and deletes them,
unintentionally creating a "pile-up error" that
discriminates against areas of high fluorophor
density.) And, in systems where slight
astigmatism has been intentionally introduced to
code for small changes in Z, won't some blobs
that are asymmetric because of motion be
"misplaced"?

At a more extreme level, we are only occasionally
interested in the location or motion of a
fluorescent molecule itself. Usually such a
molecule is used to tag some macromolecule
through a linker or even an antibody. In normal
LM, the optical spatial resolution is so poor
that displacements between fluorophor and
macromolecule are undetectable. Is this true when
the alleged resolution approaches nanometers?

On top of this, in STORM, we have the possibility
that some fluors may be turned on, and off and
then on again. In other words, not all detected
and centroided flashes need represent different
molecules. Should this affect how we view the
results? Has anyone checked for this sort of
double-dipping?

Now I am sure that most of you are already aware
that these factors exist and that they may have
some effect on the final image, but I think that
as PALM/STORM becomes more commonly used, we need
to keep in mind that the unparalleled spatial
resolution of these techniques comes at a price
and that part of this price is that many of the
comforting assumptions that simplify the
interpretation of micrographs made using more
common types of microscopy (Such as: "The
brightness of the image is proportional to the
number of tagged molecules in each location on
the specimen.") may no longer be valid.

Of the above list , I think that the most
important "noise" limitation is the small number
of single-fluorophor "flashes" detected, located
and logged into the image. It is a pity that we
can't display PALM/STORM data not as little blobs
that seem to vary in size and intensity and are
rendered in shades of yellow, orange and black
using the charming "Autumn" LUT, but in such a
way that the color of the displayed pixel makes
evident the number of fluorophors actually
detected within "x" nm of the center of each
pixel (Because of overlap, this number need not
be an integer as it may contain fractional
contributions from many nearby detection events.).

I think that such images might be quite sobering.

Happy 2012

Jim Pawley

***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953              
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ 
Application deadline 3/16/2012
               "If it ain't diffraction, it must be statistics." Anon. 11/16/12

>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello Christophe,
>As Hendrik has pointed out, there are issues with using the Thompson formula
>and this has been investigated by us and others.
>
>The rigorous approach to calculate the localization accuracy that is based
>on the Fisher information matrix was introduced by our group many years ago
>(e.g, see Ober et al., Biophys. J. 2004, 86:1185-1200, Ram et al., Proc.
>SPIE, 2005, 5699: 426-435, Ram et al., Biophys J. 2008, 95: 6025-6043).
>
>We have a software package to calculate the limit of the localization
>accuracy for different image profiles. Please check out our lab webpage for
>more info (http://www4.utsouthwestern.edu/wardlab/fandplimittool.asp).
>
>Regards,
>Sripad
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Hendrik Deschout
>Sent: Tuesday, January 24, 2012 11:08 AM
>To: [hidden email]
>Subject: Re: localization precision in PALM/STORM
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello Christophe,
>
>If your camera does not show photon counts, you can easily determine the
>conversion factor between the pixel intensity and the number of photons
>yourself, as mentioned by Andrew. The approach exploits the Poisson
>statistics of the photon numbers, see also "Scientific Charge-Coupled
>Devices" by Janesick.
>
>The interpretation of the background term "b" in the Thompson formula is
>somewhat tricky. Someone once pointed out to me that, in case you
>approximate the PSF by a two-dimensional Gaussian, it is the number of
>photons that comes from the background, plus the shoulders of the PSF which
>are not described by the Gaussian. If you again assume a Poisson
>distribution for the photon numbers, "b^2" can be determined as the variance
>of the background pixel intensities near the PSF (corrected with the
>conversion factor of course).
>
>By the way, I would like to note that the Thompson formula (and in
>particular the 30% error) has been corrected by Mortensen et al. in their
>very nice Nature Methods paper (2010, vol. 7, no. 5). Also note that the
>Thompson formula was derived for a CCD camera. In case of an electron
>multiplying CCD camera, the entire expression for "sigma^2" should be
>multiplied by 2, the electron multiplication is to blame, this is often
>ignored. In this case, it also means that the background variance you
>measure is actually equal to "2*b^2".
>
>With kind regards,
>
>Hendrik
>
>Hendrik Deschout
>Biophotonic Imaging Group
>Lab. General Biochemistry and Physical Pharmacy
>Ghent University
>Harelbekestraat 72
>9000 Gent
>Belgium
>Tel: [phone]
>Fax: [phone]
>[hidden email]
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Andrew York
>Sent: dinsdag 24 januari 2012 16:39
>To: [hidden email]
>Subject: Re: localization precision in PALM/STORM
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Expose your camera to different constant, uniform light levels, perhaps
>with a brightfield lamp. Take a bunch of pictures at each light level. For
>a given pixel, plot variance in signal vs. mean signal at each light level.
>The shape of this curve should tell you how many A/D counts you get per
>photoelectron, since photoelectron counting is hopefully a Poisson process.
>Bonus points: Do all your pixels agree on the relationship between variance
>and mean?
>
>Devil's advocate: suppose you do your calibration wrong. How would you ever
>tell? There ought to be a good answer for this. If there's no way you could
>ever tell you were wrong, what are you accomplishing?
>
>On Tue, Jan 24, 2012 at 9:51 AM, Christian Soeller <[hidden email]
>>  wrote:
>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  All cameras effectively run in a photon counting mode (more precisely they
>>  count the photo-electrons). The sCMOS cams basically have no EM gain but
>>  low enough read-out noise that it is not strictly necessary with typical
>>  photon counts. We have briefly tested a Andor Neo and it seems to work.
>>
>>  Christian
>>
>>  On 25/01/2012, at 3:29 AM, Roger Phillips wrote:
>>
>>  > The new scientific CMOS cameras are touted as applicable to single
>>  molecule localization microscopy.  But I find no mention of a photon
>>  counting mode.  How are photon counts made with these cameras?
>>  > Roger
>>  >
>>  > Dr Roger Guy Phillips
>>  > Centre for Advanced Microscopy,
>>  > University of Sussex
>>  > School of Life Sciences
>>  > John Maynard Smith Building
>>  > Falmer, Brighton & Hove
>>  > BN1 9QG
>>  > United Kingdom
>>  >
>>  > phone:[phone]
>>  > fax: [phone]
>>  > email: [hidden email]
>>  > room:2C9 (ext 7585)/lab 4C2 (ext 2734)
>>  >
>>  >
>>  >
>>  >
>>  > -----Original Message-----
>>  > From: Confocal Microscopy List [mailto:[hidden email]]
>>  On Behalf Of Christophe Leterrier
>>  > Sent: 24 January 2012 13:04
>>  > To: [hidden email]
>>  > Subject: Re: localization precision in PALM/STORM
>>  >
>>  > *****
>>  > To join, leave or search the confocal microscopy listserv, go to:
>>  > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  > *****
>>  >
>>  > Hi Guy,
>>  >
>>  > Not sure I understand what you mean here, I don't think I'm using
>>  "photon count" in my experiment. It's a wide field setup and I'm acquiring
>>  streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz
>>  framerate, with a magnified pixel size around 100 nm. The conditions are
>>  optimized for acquiring a whole photon burst (500-3000 photons depending
>on
>>  the fluorophore) in one, max two images, spread on a few pixels, to have a
>>  good signal.
>>  >
>>  > Do I have to ask Photometrics for a calibration curve between intensity
>>  levels and photoelectrons (and relate to photons using the quantum
>>  efficiency curve) ? I couldn't find that info on their website.
>>  >
>>  > Thanks for your help,
>>  >
>>  > Christophe
>>  >
>>  >
>>  > Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :
>>  >> Generally one records PALM / STORM images in photon counting mode. That
>>  means that signals below a certain threshold are regarded as noise, and
>>  discarded, and signals above a higher threshold are regarded as 'pile-up'
>>  and also discarded. So there should be no noise level to worry about and
>>  each individual image should be classed by your software as a 0 (no
>photon)
>>  or a 1 (a photon). The number of counts for that one photon, in a single
>>  frame, are meaningless. To estimate the photon counts for a point in the
>>  image you need to see in how many frames that point is scored as a 1. I
>>  hope this makes sense!
>>  >>
>>  >> Guy
>>  >>
>>  >> Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press /
>>  >> Taylor & Francis http://www.guycox.com/optical.htm
>>  >> ______________________________________________
>>  >> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
>>  >> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
>>  >> NSW 2006
>>  >>
>>  >> Phone [phone] Fax [phone] Mobile [phone]
>>  >> ______________________________________________
>>  >> http://www.guycox.net
>>  >>
>>  >>
>>  >> -----Original Message-----
>>  >> From: Confocal Microscopy List
>>  >> [mailto:[hidden email]] On Behalf Of Christophe
>>  >> Leterrier
>>  >> Sent: Tuesday, 24 January 2012 8:27 PM
>>  >> To: [hidden email]
>  > >> (mailto:[hidden email])
>>  >> Subject: localization precision in PALM/STORM
>>  >>
>>  >> *****
>>  >> To join, leave or search the confocal microscopy listserv, go to:
>>  >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  >> *****
>>  >>
>>  >> Hi,
>>  >>
>>  >> Not strictly a confocal question, but I'm pretty sure this list is the
>>  best place to get thorough and insightful answers.
>>  >>
>>  >> I have made 2D STORM (stochastic optical reconstruction microscopy)
>>  acquisitions and processing and I end up with a table of XY localized
>>  fluorophores together with the integrated intensity of the localized
>>  diffraction-limited spot.
>>  >>
>>  >> I'd like to plot each fluorophore as a gaussian with a width
>>  corresponding to the localization precision, similar to what was done in
>>  Bates et al. Science 2007. According to equation (17) in Thompson, Larson
>&
>>  Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the
>>  number of photons collected, the width of the diffraction-limited spot,
>the
>>  size of the camera pixel, and the background noise.
>>  >>
>>  >> So my question is : How do I get the number of photons from the
>>  intensity level of an image? I'm using a Photometrics 512*512 QuantEM
>>  camera. What is the background noise and how do I estimate it? Then using
>>  these values in the Thompson et al. equation, I can get a theoretical spot
>>  intensity / localization precision calibration curve that I could use for
>>  the gaussian-based reconstruction.
>>  >>
>>  >> Thanks for your help,
>>  >>
>>  >> --
>>  >> Christophe Leterrier
>>  >> Researcher
>>  >> Axonal Domains Architecture Team
>>  >> CRN2M CNRS UMR 7286
>>  >> Aix Marseille University, France
>>  >>
>>  >>
>>  >>
>>  >>
>>
>>  --
>>  Christian Soeller PhD   Dept. of Physiology  [phone] x82770
>>  University of Auckland  Auckland, New Zealand  fax [phone]
>>

--
***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953              
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ 
Application deadline 3/16/2012
               "If it ain't diffraction, it must be statistics." Anon. 11/16/12

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I just assumed that photon-counting technology would be used for this but I guess I was wrong.  I also thought astronomers used CCD and CMOS sensors for photon counting.  I guess it depends on how many electrons one photon produces in the potential well.  Jim probably knows the answer to that.

                               Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Soeller
Sent: Wednesday, 25 January 2012 2:30 AM
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Essentially yes.

Guy's reply would seem to apply to photon-counting with PMTs when run in photon-counting-mode, not to cameras. Guy, please correct me if I am wrong.

Christian

On 25/01/2012, at 4:18 AM, Roger Phillips wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks, Christian,
> So the sCMOS photon count would come from the formula
> photons = (counts - A/D offset)*electrons-per-count/EM-gain
> with EM-gain set to 1 and no 'additional noise introduced by the EM gain process'?
>
> In Guy's reply, he said 'signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded.'  This methods seems to account for the 'low threshold' but not for the possible 'pile-up'?
> Roger
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Soeller
> Sent: 24 January 2012 14:44
> To: [hidden email]
> Subject: Re: localization precision in PALM/STORM
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> You basically want to convert the AD counts in your image into photon numbers. That requires several bits of info about the camera. We have Andor cameras and I can look up the required values from the data/test sheet that comes with the individual camera (these change from cam to cam). You need to know the
> - electrons per count
> - absolute EM gain (with the quantEM you might have to measure/calibrate this)
> - A/D offset (this can be measured in dark frames with no light impinging on the cam, i.e. shutter closed)
>
> The formula is then something like
>
> photons = (counts - A/D offset)*electrons-per-count/EM-gain
>
> Some more recent camera software packages may have functions to make this conversion for you. I am not sure about photometrics cams/SDKs.
>
> Due the additional noise introduced by the EM gain process you should divide the resulting photon-numbers by 2 before looking up values from the Thompson et al. formula. There are papers on EM CCDs that explain this. I also seem to recall that some more recent papers have a subtle correction to the Thompson formula.
>
> Hope this helps,
>
> Christian
>
> On 25/01/2012, at 2:08 AM, Christophe Leterrier wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Mark,  
>>
>> I don't think the quantEM has a built-in photon calibration function, in contrast to the newer Evolve camera, also from Photometrics. The spec sheet for the quantEM is available here :
>> http://www.photometrics.com/products/datasheets/qem512sc.pdf
>>
>> Do I have to calibrate it myself or is Photometrics supposed to provide a photon/intensity calibration curve? I don't want exact experimental values for my precise camera, just a reasonable estimate to derive a theoretical "best" value for localization accuracy.
>>
>> Christophe  
>>
>>
>> Le mardi 24 janvier 2012 à 10:58, Mark Cannell a écrit :
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Doesn't the quantEM have a photon calibration function? The background noise should be estimated from the variance of the background (extracted from image regions when/where flashes were not detected...). You can also calibrate the camera with weak sources to double check the manufactures stated read-out calibration.
>>>
>>> Hope this helps
>>>
>>> Mark
>>>
>>>
>>> On 24/01/2012, at 9:26 AM, Christophe Leterrier wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi,  
>>>>
>>>> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>>>>
>>>> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot.  
>>>>
>>>> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>>>>
>>>> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>>>>
>>>> Thanks for your help,
>>>>
>>>> --
>>>> Christophe Leterrier
>>>> Researcher
>>>> Axonal Domains Architecture Team
>>>> CRN2M CNRS UMR 7286
>>>> Aix Marseille University, France
>>>>
>>>
>>>
>>>
>

[Alex Knight]

James Pawley wrote

However, while it is certainly important to know noise levels of the various photodetectors, there may be other noise sources that may be even more important.

What about the statistical and other errors related to the very small numbers of fluorescent molecules actually localized?

Hi James, you make a lot of excellent points here, and ones that it is easy to forget about.

One could add to your list:
- what about the molecules that you never see because they don't blink during your acquisition, or photobleach first?
- what about overlapping molecules which are not rejected by the software, but give rise to mis-localisations?
- worst of all, what about mechanical drift occurring in the system during the acquisition? If this is not controlled, it can seriously degrade your images.

I could go on. Really the importance of all these various factors depends on the biological question you are trying to address and the type of sample you are using.

The fluorophore orientation issue you raise is interesting, but I think that in most situations the fluorophores rotate freely enough that polarisation is not a problem.

Cheers

Alex

PS For those of you who are on LinkedIn we have just set up a super-resolution/nanoscopy group which may be of interest.

Alex Knight, National Physical Laboratory

[Andreas Bruckbauer]

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 Hi,
back to the original question, i would say that because of the effects of stage drift and unknowns such as the quality of the fitting routines it is best to measure the localisation precision directly. In Storm you should be able get several localisations from individual molecules and get an average localistion precision out of these (fit to a histogram). You can do this for your label immobilised on a glass slide at very low densities and for fixed cells. The later case should give a slightly lower localisation precision because of the higher background noise. If these localisation precision does not vary much in the sample and between experiments, you can construct the images using these average values.

If the molecule you are labelling is abundand enough and the width of the biological structures small enough, you can measure this width in your images and compare with the estimated localisation precision, i think this ahould be the ultimate test if what you are doing is reasonable (as shown in the case of tubulin in many papers).

Furthermore, if you have bright labels, the signal to noise ratio should be high and the localisation precision dominated by the shot noise of the peak and not the background noise. This can be quite different from the case of single molecule tracking at video rates where you get much lower signal to noise ratios.

best wishes

Andreas

 

 

-----Original Message-----
From: Christophe Leterrier <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Tue, 24 Jan 2012 9:53
Subject: localization precision in PALM/STORM


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Hi,



Not strictly a confocal question, but I'm pretty sure this list is the best

place to get thorough and insightful answers.



I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions

and processing and I end up with a table of XY localized fluorophores together

with the integrated intensity of the localized diffraction-limited spot.



I'd like to plot each fluorophore as a gaussian with a width corresponding to

the localization precision, similar to what was done in Bates et al. Science

2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002

(http://goo.gl/5GIXM), this precision depends on the number of photons

collected, the width of the diffraction-limited spot, the size of the camera

pixel, and the background noise.



So my question is :  How do I get the number of photons from the intensity level

of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the

background noise and how do I estimate it? Then using these values in the

Thompson et al. equation, I can get a theoretical spot intensity / localization

precision calibration curve that I could use for the gaussian-based

reconstruction.



Thanks for your help,



--

Christophe Leterrier

Researcher

Axonal Domains Architecture Team

CRN2M CNRS UMR 7286

Aix Marseille University, France







 



 


 

[David Baddeley]

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Photon counting mode on EMCCDs  works by making the em gain very high, such that the read noise is much less than 1 photon per pixel. If the photon flux is also very low (much less than 1 photon/pixel/frame) you can threshold each frame and classify each pixel as either having seen a photon or not, and thereby remove the multiplicative noise effects. This only works for very low photon counts as are observed in some aspects of astronomy. In PALM/STORM the photon fluxes are much higher (typically several 100-1000 /pixel/frame) and we can't do photon counting per se. As to EMCCD vs CCD vs SCMOS, they all work with a slight theoretical advantage to SCMOS followed closely by EMCCD. In most practical cases the background is sufficiently high that a small amount of read noise can be tolerated (even as much as the ~5 e- you get from a good conventional CCD @ ~10hz readout - although you'll often want to record faster than 10 hz at which point standard ccds
 become less useful). The important factors for PALM/STORM are thus effective quantum efficiency (remembering that the electron multiplying step effectively halves the QE), and readout speed.  I remember hearing that Leica was using a sCMOS device in their system (they've definitely published a proof of concept with one), and when we trialled a CMOS sensor it seemed to work just as well as our EMCCD cameras.

As to orientation effects, I was unable to see any difference between linearly and circularly polarised excitation when imaging using antibody conjugated dyes - this suggests that they (at least) are on a flexible linker and free to rotate rapidly. Can't really speculate as to how well this extends to other labelling methods.

I'd also suggest investigating other visualisation methods, as the original method of drawing Gaussians has a number of potential limitations, some of which, such as the tendency to inspire more confidence in the data than might be warranted, have already been pointed out (what hasn't been mentioned yet is the fact that it sacrifices resolution if you do happen to have a high density of localisations).  Try a number of methods and see how they work with your data (and ideally also with simulated data of known objects). At the very least do a simple 2D histogram as well as the Gaussian method - this will allow you to directly relate pixel intensity to the number of events within that pixel. If you want to go further, our paper (Baddeley, Cannell, Soeller, 'Visualization of Localization Microscopy Data', Microscopy and Microanalysis 2010) might give you some ideas.

Best wishes,
David


________________________________
 From: Guy Cox <[hidden email]>
To: [hidden email]
Sent: Wednesday, 25 January 2012 11:20 AM
Subject: Re: localization precision in PALM/STORM
 
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I just assumed that photon-counting technology would be used for this but I guess I was wrong.  I also thought astronomers used CCD and CMOS sensors for photon counting.  I guess it depends on how many electrons one photon produces in the potential well.  Jim probably knows the answer to that.

                               Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Soeller
Sent: Wednesday, 25 January 2012 2:30 AM
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

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Essentially yes.

Guy's reply would seem to apply to photon-counting with PMTs when run in photon-counting-mode, not to cameras. Guy, please correct me if I am wrong.

Christian

On 25/01/2012, at 4:18 AM, Roger Phillips wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thanks, Christian,
> So the sCMOS photon count would come from the formula
> photons = (counts - A/D offset)*electrons-per-count/EM-gain
> with EM-gain set to 1 and no 'additional noise introduced by the EM gain process'?
>
> In Guy's reply, he said 'signals below a certain threshold are regarded as noise, and discarded, and signals above a higher threshold are regarded as 'pile-up' and also discarded.'  This methods seems to account for the 'low threshold' but not for the possible 'pile-up'?
> Roger
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Soeller
> Sent: 24 January 2012 14:44
> To: [hidden email]
> Subject: Re: localization precision in PALM/STORM
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> You basically want to convert the AD counts in your image into photon numbers. That requires several bits of info about the camera. We have Andor cameras and I can look up the required values from the data/test sheet that comes with the individual camera (these change from cam to cam). You need to know the
> - electrons per count
> - absolute EM gain (with the quantEM you might have to measure/calibrate this)
> - A/D offset (this can be measured in dark frames with no light impinging on the cam, i.e. shutter closed)
>
> The formula is then something like
>
> photons = (counts - A/D offset)*electrons-per-count/EM-gain
>
> Some more recent camera software packages may have functions to make this conversion for you. I am not sure about photometrics cams/SDKs.
>
> Due the additional noise introduced by the EM gain process you should divide the resulting photon-numbers by 2 before looking up values from the Thompson et al. formula. There are papers on EM CCDs that explain this. I also seem to recall that some more recent papers have a subtle correction to the Thompson formula.
>
> Hope this helps,
>
> Christian
>
> On 25/01/2012, at 2:08 AM, Christophe Leterrier wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Mark, 
>>
>> I don't think the quantEM has a built-in photon calibration function, in contrast to the newer Evolve camera, also from Photometrics. The spec sheet for the quantEM is available here :
>> http://www.photometrics.com/products/datasheets/qem512sc.pdf
>>
>> Do I have to calibrate it myself or is Photometrics supposed to provide a photon/intensity calibration curve? I don't want exact experimental values for my precise camera, just a reasonable estimate to derive a theoretical "best" value for localization accuracy.
>>
>> Christophe 
>>
>>
>> Le mardi 24 janvier 2012 à 10:58, Mark Cannell a écrit :
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Doesn't the quantEM have a photon calibration function? The background noise should be estimated from the variance of the background (extracted from image regions when/where flashes were not detected...). You can also calibrate the camera with weak sources to double check the manufactures stated read-out calibration.
>>>
>>> Hope this helps
>>>
>>> Mark
>>>
>>>
>>> On 24/01/2012, at 9:26 AM, Christophe Leterrier wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi, 
>>>>
>>>> Not strictly a confocal question, but I'm pretty sure this list is the best place to get thorough and insightful answers.
>>>>
>>>> I have made 2D STORM (stochastic optical reconstruction microscopy) acquisitions and processing and I end up with a table of XY localized fluorophores together with the integrated intensity of the localized diffraction-limited spot. 
>>>>
>>>> I'd like to plot each fluorophore as a gaussian with a width corresponding to the localization precision, similar to what was done in Bates et al. Science 2007. According to equation (17) in Thompson, Larson & Webb Biophys J. 2002 (http://goo.gl/5GIXM), this precision depends on the number of photons collected, the width of the diffraction-limited spot, the size of the camera pixel, and the background noise.
>>>>
>>>> So my question is : How do I get the number of photons from the intensity level of an image? I'm using a Photometrics 512*512 QuantEM camera. What is the background noise and how do I estimate it? Then using these values in the Thompson et al. equation, I can get a theoretical spot intensity / localization precision calibration curve that I could use for the gaussian-based reconstruction.
>>>>
>>>> Thanks for your help,
>>>>
>>>> --
>>>> Christophe Leterrier
>>>> Researcher
>>>> Axonal Domains Architecture Team
>>>> CRN2M CNRS UMR 7286
>>>> Aix Marseille University, France
>>>>
>>>
>>>
>>>
>