Posted by
Crowell Elizabeth on
Mar 10, 2011; 12:40pm
URL: http://imagej.273.s1.nabble.com/Deconvolution-on-MacBook-tp3685439p3685442.html
Dan White a écrit :
> Dear Elizabeth,
> Sure, why not contact the authors of the plugin directly... that are human and will respond to well formed questions.
> I have met them, and they are good folks as well as experts in the methods.
>
Yes, I have contacted them as well, but it is always interesting to get
input from users as well as the developers!
>> I would like to try calculating a theoretical PSF. There is some ambiguity in the parameters to input into the PSF generator from the BIG lab. For example, is "refractive index" the r.i. of the lens objective medium, or the sample mounting medium?
>>
>
> 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=pluginsThe documentation amounts to: Select the parameters and click on
"Generate PSF". I am not joking! So I contacted the author with my
specific questions.
> this is the pixel separation in x y and z no?
> You can only roughly calculate it from the camera pixel size and the nominal magnification of the system. This might be wrong by 10% or more.
> Best to get a stage micrometer and measure it!
>
Okay, I have done this already.
>> Is the "wavelength" the excitation or emission wavelength?
>>
> emission.
>
>> 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.
> 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.
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
>
>
>