Re: localization precision in PALM/STORM

Posted by Roger Phillips on
URL: http://confocal-microscopy-list.275.s1.nabble.com/localization-precision-in-PALM-STORM-tp7219699p7220543.html

The new scientific CMOS cameras are touted as applicable to single molecule localization microscopy.  But I find no mention of a photon counting mode.  How are photon counts made with these cameras?
Roger

Dr Roger Guy Phillips
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: 24 January 2012 13:04
To: [hidden email]
Subject: Re: localization precision in PALM/STORM

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Hi Guy,  

Not sure I understand what you mean here, I don't think I'm using "photon count" in my experiment. It's a wide field setup and I'm acquiring streams of EMCCD camera full-frame (512px*512px) images at 10-20 Hz framerate, with a magnified pixel size around 100 nm. The conditions are optimized for acquiring a whole photon burst (500-3000 photons depending on the fluorophore) in one, max two images, spread on a few pixels, to have a good signal.

Do I have to ask Photometrics for a calibration curve between intensity levels and photoelectrons (and relate to photons using the quantum efficiency curve) ? I couldn't find that info on their website.

Thanks for your help,

Christophe


Le mardi 24 janvier 2012 à 12:06, Guy Cox a écrit :  

> Generally one records PALM / STORM images in photon counting mode. That means that 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. So there should be no noise level to worry about and each individual image should be classed by your software as a 0 (no photon) or a 1 (a photon). The number of counts for that one photon, in a single frame, are meaningless. To estimate the photon counts for a point in the image you need to see in how many frames that point is scored as a 1. I hope this makes sense!
>  
> Guy
>  
> Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press /
> Taylor & Francis http://www.guycox.com/optical.htm 
> ______________________________________________
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> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Christophe
> Leterrier
> Sent: Tuesday, 24 January 2012 8:27 PM
> To: [hidden email]
> (mailto:[hidden email])
> Subject: localization precision in PALM/STORM
>  
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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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