http://confocal-microscopy-list.275.s1.nabble.com/localization-precision-in-PALM-STORM-tp7219699p7222213.html
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'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.
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> 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
>
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> 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:
>
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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 :
>>
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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:
>>>
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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
>>>>
>>>
>>>
>>>
>