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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 <[log in to unmask]>
To: [log in to unmask] 
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 +61 2 9351 3176     Fax +61 2 9351 7682
             Mobile 0413 281 861
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      http://www.guycox.net



-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Christian Soeller
Sent: Wednesday, 25 January 2012 2:30 AM
To: [log in to unmask]
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:[log in to unmask]] On Behalf Of Christian Soeller
> Sent: 24 January 2012 14:44
> To: [log in to unmask]
> 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
>>>> 
>>> 
>>> 
>>> 
>