Posted by
Daniel James White on
Mar 09, 2011; 9:13pm
URL: http://imagej.273.s1.nabble.com/Deconvolution-on-MacBook-tp3685439p3685441.html
Dear Elizabeth,
I thought I would cc this to the list, to share the words... hope that's ok.
On Mar 9, 2011, at 8:45 PM, Crowell Elizabeth wrote:
> Dear Dr. White,
>
> Thank you very much for sharing some very useful information on the ImageJ mailing list about deconvolution.
> I was happy to have your recommendation for using Deconvolution Lab. It is indeed often difficult to choose a plugin from the many ones available!
> Although Deconvolution Lab looks promising for my purposes, the webpage gives minimal information on use. I am not experienced enough to understand everything, I'm afraid! Would you mind giving me a little advice?
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.
>
> 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.
> How do I calculate the spatial and axial resolution?
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!
point scanning confocals are spatially calibrated and record the pixel separation in the meta data of the files they save, but scanners should also be calibrated,
> 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.
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!
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)
>
> I realize these must sound like very naive questions,
not at all...
> but it is the first time I try to calculate a PSF and I would like to understand what I am doing. I hope you will be kind enough to answer, since I have some trouble finding help so far! I have spent quite some time reading the excellent website of SVI, but my questions remain unfortunately unanswered.
feel free to ask more on the imagej list...
cheers
Dan
>
> Kind Regards,
>
> Elizabeth Crowell
>
>
>
>
>
> Daniel James White a écrit :
>> Hi Mark,
>>
>> On Mar 9, 2011, at 6:00 AM, IMAGEJ automatic digest system wrote:
>>
>>
>>
>>> Date: Tue, 8 Mar 2011 11:42:17 -0500
>>> From: "Mark Adelman (Work)"
>>> <
[hidden email]>
>>>
>>> Subject: Deconvolution on MacBook?
>>>
>>> Is it realistic to attempt to do a limited number of wide field
>>> deconvolutions using ImageJ on a MacBook?
>>>
>>>
>>
>> Yes, so long as the images are small enough to fit in your RAM with a copy or two also in RAM...
>> So RAM needs to be a few times larger than the size of the image.
>>
>>
>>
>>> My MacBook has a 2.13 GHz
>>> Intel Core 2 Duo Processor and Memory of 2 GB 800 MHz DDR2 SDRAM.
>>>
>>>
>>
>> so the biggest image you could probably try and deconvolve in imageJ is limited by the
>> 2GB ram, less whatever the MacOSX is usiung for itself...in the ideal case.
>>
>> Close all other applications and reboot the computer,
>> and only open imageJ or Fiji,
>> so the java VM can get that whole nearly 2GB RAM in a single continuous chunk.
>>
>> If the memory is already fragmented in to smaller bits, you will struggle.
>>
>> Try DeconvolutionLab plugin from the BIG group in lausanne.
>> Its very good.
>>
>>
http://bigwww.epfl.ch/algorithms/deconvolutionlab/>>
>>
>> Use a measured PSF (image og a 100 nm bead z stack)
>> or use Bob's Diffraction PSF 3D plugin to calculate a pretend 3D widefield PSF
>> and use that in the deconvolution.
>>
>>
>>
http://www.optinav.com/Diffraction-PSF-3D.htm>>
>>
>>
>>
>>
>>> I
>>> am running Leopard (OS 10.5.8). I have the ability to capture Z
>>> series fluorescence microscopy data sets (Zeiss Photomicroscope III
>>>
>>>
>>
>> thats a very cool old microscope!
>>
>>
http://www.microscopy-uk.org.uk/mag//artnov07/dw-pm3.html>>
>>
>> How are you doing fluorescence on it? II dint see where the filter sets go? Do you have the epi accessories?
>>
>> Deconvolution doesnt really work on transmitted light images... there are approaches for that... but
>> the tools above are strictly for fluorescence.
>>
>>
>>
>>
>>> with a Focus Controller and a digital camera), under the control of an
>>> iMac (running under 10.4.11). I already know the iMac does not have
>>> enough computing power. If anyone has suggestions regarding
>>> possibilities using the MacBook, please contact me offline (unless you
>>> think others would be interested).
>>>
>>>
>>
>> the mac book will work fine... so long as the java VM doesnt run out of RAM.
>>
>> so for small images (say 256x256x20) you might be fine,
>> but large images, eg full camera chip image and many z slices you will be in trouble...
>>
>> then the only solution is a powerful 64 bit workstation with tens of GB or ram and multiple processors.
>>
>> you need to make sure that the magnification is matched to the camera xy pixel spacing so that you
>> fulfill the Nyquist sampling criterion, and that you z slice separation is also fulfilling that criterion, and its precise.
>>
>> What objective are you using?
>>
>> you can calculate the xyz pixel sizes needed to get data suitable doe deconvolution here:
>>
>>
http://www.svi.nl/NyquistCalculator>>
>> and read up more on that wiki, it is excellent for understanding how to do deconvolution microscopy.
>>
>>
>>
>>
>>> Thanks in advance!!
>>>
>>>
>>
>> but I didn't help you before I wrote this email...
>>
>> if you like you can retract the premature felicitations, and thank me now instead!!
>>
>> cheers
>>
>> Dan
>>
>>
>>
>>> Mark R. Adelman
>>>
>>>
[hidden email]
>>>
>>>
>>>
>>
>> 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
>> Pfotenhauerstrasse 108
>> 01307 DRESDEN
>> Germany
>>
>> +49 (0)15114966933 (German Mobile)
>> +49 (0)351 210 2627 (Work phone at MPI-CBG)
>> +49 (0)351 210 1078 (Fax MPI-CBG LMF)
>>
>>
>>
http://www.bioimagexd.net>> BioImageXD
>>
>>
http://pacific.mpi-cbg.de>> Fiji - is just ImageJ (Batteries Included)
>>
>>
http://www.chalkie.org.uk>> Dan's Homepages
>>
>>
https://ifn.mpi-cbg.de>> Dresden Imaging Facility Network
>> dan (at) chalkie.org.uk
>> ( white (at) mpi-cbg.de )
>>
>>
>
>
> --
>
> Elizabeth CROWELL
>
> ----------------------------------------------------------------------
> Membrane Traffic and Cell Division Research Group
> Institut Pasteur
> 28 rue du Dr Roux
> 75015 PARIS, France
>
> Tel : 01.44.38.94.07
> Fax : 01.45.68.89.54
> ----------------------------------------------------------------------
>
Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Processing and Analysis
Light Microscopy Facility
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany
+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
+49 (0)351 210 1078 (Fax MPI-CBG LMF)
http://www.bioimagexd.net BioImageXD
http://pacific.mpi-cbg.de Fiji (is just ImageJ - batteries included)
http://www.chalkie.org.uk[hidden email]
(
[hidden email] )