http://confocal-microscopy-list.275.s1.nabble.com/localization-precision-in-PALM-STORM-tp7219699p7220306.html
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 :
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.
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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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