Role FWHM and Pixel Size during the image reconstruction

Bayesian analysis of blinking and bleaching

Role FWHM and Pixel Size during the image reconstruction

Postby william2014 » Wed Dec 17, 2014 2:51 pm

Hi there,

i have 3 questions concerning the influence of the blur FWHM and pixel Size during the image reconstruction with 3B using the imageJ plugin:

- What are the role of the blur FWHM and pixel size during the reconstruction? If i change them at any stage of the reconstruction, can this affect my reconstruction?
- At the beginning of the programm, how should i set the Blur FHWM and the pixel size, if i want to obtain a very good image reconstruction ( in the 3b help Tutorial, blur=16.1 nm and pixel= 3.0 nm).
- If the Blur FHWM and the number of Pixel are small, the programm will run slow of fast?

Thanks
william2014
 
Posts: 3
Joined: Thu Nov 13, 2014 10:20 am

Re: Role FWHM and Pixel Size during the image reconstruction

Postby edrosten » Thu Dec 18, 2014 5:40 pm

william2014 wrote:Hi there,
- What are the role of the blur FWHM and pixel size during the reconstruction? If i change them at any stage of the reconstruction, can this affect my reconstruction?


The reconstruction after analysis is pretty much the same for 3B as for every other localisation microscopy technique. Localisation techniques generate a list of fluorophore positions. Do reconstruct, these are drawn on to a pixel grid and then blurred slightly.



- At the beginning of the programm, how should i set the Blur FHWM and the pixel size, if i want to obtain a very good image reconstruction ( in the 3b help Tutorial, blur=16.1 nm and pixel= 3.0 nm).


The reconstruction blur and pixel size are independent of the analysis. The quality of the reconstruction will depend on these two. Generally you want the blur size to be small enough to capture the super resolved features, but large enough so that you don't get artefacts due to sampling. The pixel size needs to be a fair bit smaller than the FWHM: if the pixel size is too large then the image will look pixellated. You probably want the pixel size to be somewhere around 1/4 to 1/10 of the blur FWHM in order to avoid visible pixellation.


- If the Blur FHWM and the number of Pixel are small, the programm will run slow of fast?


Very small pixels sizes and very large blurs will run more slowly that if more moderate values are used.

Regards

-Ed
edrosten
 
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Joined: Wed Jul 18, 2012 2:54 pm


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