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Hello Christophe,
If your camera does not show photon counts, you can easily determine the conversion factor between the pixel intensity and the number of photons yourself, as mentioned by Andrew. The approach exploits the Poisson statistics of the photon numbers, see also "Scientific Charge-Coupled Devices" by Janesick.
The interpretation of the background term "b" in the Thompson formula is somewhat tricky. Someone once pointed out to me that, in case you approximate the PSF by a two-dimensional Gaussian, it is the number of photons that comes from the background, plus the shoulders of the PSF which are not described by the Gaussian. If you again assume a Poisson distribution for the photon numbers, "b^2" can be determined as the variance of the background pixel intensities near the PSF (corrected with the conversion factor of course).
By the way, I would like to note that the Thompson formula (and in particular the 30% error) has been corrected by Mortensen et al. in their very nice Nature Methods paper (2010, vol. 7, no. 5). Also note that the Thompson formula was derived for a CCD camera. In case of an electron multiplying CCD camera, the entire expression for "sigma^2" should be multiplied by 2, the electron multiplication is to blame, this is often ignored. In this case, it also means that the background variance you measure is actually equal to "2*b^2".
With kind regards,
Hendrik
Hendrik Deschout
Biophotonic Imaging Group
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Gent
Belgium
Tel: +32 9 264.80.74
Fax: +32 9 264.81.89
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Andrew York
Sent: dinsdag 24 januari 2012 16:39
To: [log in to unmask]
Subject: Re: localization precision in PALM/STORM
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Expose your camera to different constant, uniform light levels, perhaps
with a brightfield lamp. Take a bunch of pictures at each light level. For
a given pixel, plot variance in signal vs. mean signal at each light level.
The shape of this curve should tell you how many A/D counts you get per
photoelectron, since photoelectron counting is hopefully a Poisson process.
Bonus points: Do all your pixels agree on the relationship between variance
and mean?
Devil's advocate: suppose you do your calibration wrong. How would you ever
tell? There ought to be a good answer for this. If there's no way you could
ever tell you were wrong, what are you accomplishing?
On Tue, Jan 24, 2012 at 9:51 AM, Christian Soeller <[log in to unmask]
> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> > Falmer, Brighton & Hove
> > BN1 9QG
> > United Kingdom
> >
> > phone:44 (0)1273 877585
> > fax: 44 (0)1273 678433
> > email: [log in to unmask]
> > room:2C9 (ext 7585)/lab 4C2 (ext 2734)
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Christophe Leterrier
> > Sent: 24 January 2012 13:04
> > To: [log in to unmask]
> > Subject: Re: localization precision in PALM/STORM
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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
> >>
> >> Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861
> >> ______________________________________________
> >> http://www.guycox.net
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >> [mailto:[log in to unmask]] On Behalf Of Christophe
> >> Leterrier
> >> Sent: Tuesday, 24 January 2012 8:27 PM
> >> To: [log in to unmask]
> >> (mailto:[log in to unmask])
> >> Subject: localization precision in PALM/STORM
> >>
> >> *****
> >> 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
>
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