Re: localization precision in PALM/STORM

Posted by Christian Soeller on
URL: http://confocal-microscopy-list.275.s1.nabble.com/localization-precision-in-PALM-STORM-tp7219699p7220589.html

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All cameras effectively run in a photon counting mode (more precisely they count the photo-electrons). The sCMOS cams basically have no EM gain but low enough read-out noise that it is not strictly necessary with typical photon counts. We have briefly tested a Andor Neo and it seems to work.

Christian

On 25/01/2012, at 3:29 AM, Roger Phillips wrote:

> 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
> Centre for Advanced Microscopy,
> University of Sussex
> School of Life Sciences
> John Maynard Smith Building
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> United Kingdom
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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 
>> ______________________________________________
>> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
>> Microscopy & Microanalysis, Madsen Building F09, University of Sydney,
>> NSW 2006
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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
>>
>>
>>
>>

--
Christian Soeller PhD   Dept. of Physiology  [phone] x82770
University of Auckland  Auckland, New Zealand  fax [phone]