Posted by
Christian Soeller on
URL: http://confocal-microscopy-list.275.s1.nabble.com/localization-precision-in-PALM-STORM-tp7219699p7220587.html
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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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>
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 +64 9 3737599 x82770
University of Auckland Auckland, New Zealand fax +64 9 3737499