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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
> 
> 
> 
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>