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 Hi,
back to the original question, i would say that because of the effects of stage drift and unknowns such as the quality of the fitting routines it is best to measure the localisation precision directly. In Storm you should be able get several localisations from individual molecules and get an average localistion precision out of these (fit to a histogram). You can do this for your label immobilised on a glass slide at very low densities and for fixed cells. The later case should give a slightly lower localisation precision because of the higher background noise. If these localisation precision does not vary much in the sample and between experiments, you can construct the images using these average values. 

If the molecule you are labelling is abundand enough and the width of the biological structures small enough, you can measure this width in your images and compare with the estimated localisation precision, i think this ahould be the ultimate test if what you are doing is reasonable (as shown in the case of tubulin in many papers). 

Furthermore, if you have bright labels, the signal to noise ratio should be high and the localisation precision dominated by the shot noise of the peak and not the background noise. This can be quite different from the case of single molecule tracking at video rates where you get much lower signal to noise ratios.

best wishes

Andreas

 

 

-----Original Message-----
From: Christophe Leterrier <[log in to unmask]>
To: CONFOCALMICROSCOPY <[log in to unmask]>
Sent: Tue, 24 Jan 2012 9:53
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