Re: Deconvolution Was: Deconvolution on MacBook

Posted by lechristophe on Mar 10, 2011; 4:15pm
URL: http://imagej.273.s1.nabble.com/Deconvolution-on-MacBook-tp3685439p3685444.html

Hi,

What I did once (I was using Volocity at the time) was to generate a virtual
PSF (you can do that with Bob Dougherty's plugin or the EPFL one), but use a
real empirical PSF from my setup to infer the real NA to feed the PSF
generation.

To do that, just measure the angle of the PSF cone in XZ, like explained
here : http://www.svi.nl/NumericalAperture

So you get the advantages of a calculated PSF (noise-free), but with an
overall shape that corresponds to your setup. Surpirisngly, the objective
was 1.3 but the measured NA from the cone anlgle was around 1.1 !

Anyway, the svi website is a great resource for all things deconvolution.

Cheers,

Christophe


On Thu, Mar 10, 2011 at 15:43, Daniel James White <[hidden email]> wrote:

> Hi Elizabeth,
> >>
> >> which plugin exactly do you mean?
> >> Indeed there are 2  RIs to take care of, and you correctly query which
> it asks for... better ask them or look for clarification in the docs.
> >>
> >>
> > I am referring to the PSF generator plugin at
> http://bigwww.epfl.ch/deconvolution//?p=plugins
> > The documentation amounts to: Select the parameters and click on
> "Generate PSF".  I am not joking!  So I contacted the author with my
> specific questions.
>
> OK, so there is that one, and also Bob's Diffraction PSD 3D plugin.
>
> They give similar but slightly different PSFs for the same input
> parameters,
> as they must use a different model to generate the image....?
> >>
> >>> I also have stacks of subresolution size beads that I would like to use
> to measure my PSF.  How can I obtain this measurement?
> >>>
> >> the image of 1 bead is pretty much almost-ish  the PSF. So long as its
> really a sub resolution bead. You can align multiple images of beads and
> average them.
> >> But if you get a single good bead with very good signal : noise, that
> will work.
> >>
> >>
> > This is a strange concept for me, that a "function" could be equated to
> an image stack!  But I suppose the mathematical function itself is
> calculated from the image stack by the software?  After all the information
> I have read, PSF still remains a vague term for me, sometimes used to
> describe an image, sometimes used to describe the diffraction pattern of
> light around an object, sometimes used to describe the mathematical function
> that will be applied to the raw image to deconvolve it.
>
> you are right, it is odd to talk about a discretized image ( a set of point
> values) as a function... its just the jargon of the field.
> Physically, it is a smooth function, but we have to deal with a discrete
> representation of it... an "image", in order to do the deconvolution.
> I understand your confusion, and it is well grounded
> >
> >> Huygens software has a tool to make a psf from a bead image by removing
> the size of the bead and cleaning up the psf image.
> >> API softWoRx does it too (OTF in this case)
> >> Not sure i saw a tool like this for imageJ... if you did then please let
> me know!
> >>
> > Yes, it is the plugin by Janick Cardinale from the Mosaic lab.  You can
> find the pdf manual at
> www.mosaic.ethz.ch/Downloads/ParticleTracker/PSF_measurement_3D.pdf
> > But I cannot find the plugin anywhere!  I wrote to the author.
>
> I has a quick look... this is not what you need, as you want to measure the
> PSF in 3D,
> but this one takes bead images and averaved them axially, to make a 2D PSF
> that is a rotational projection around the axis. You dont want that,
>
> What you want is a gadget likje the Huygens PSF distiller that makes 3D
> PSFs from real bead images....
>
> but you can use an image of a bead that is 100nm AS the PSF.
>
>
> cheers
>
> Dan
>
>
> >
> > To be continued...  thanks again,
> > Elizabeth
> >
> >
> >
> >> For use as a psf, the bead image must be centered on the centre of the
> bead for it to work as a psf.
> >>
> >>
> >>
> >>> I found information on a PSF 3D measurement plugin by Janick Cardinale,
> but it does not appear to be available for download in the list of ImageJ
> plugins, and I find no similar plugins.
> >>>
> >>>
> >>
> >> can you share that info?
> >> Also see the mosaic plugins from ETH (i haven't tested them yet though)
> >> feel free to ask more on the imagej list...
> >>
> >> cheers
> >>
> >>
> >> Dan
> >>
> >>
> >>
> >>
> >
>
> Dr. Daniel James White BSc. (Hons.) PhD
> Senior Microscopist / Image Visualisation, Processing and Analysis
> Light Microscopy and Image Processing Facilities
> Max Planck Institute of Molecular Cell Biology and Genetics
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> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
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>
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> Included)
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>