http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-3D-SIM-data-tp6251420p6268319.html
in a ideal case (subresolutive beads sufficiently spaced). Of course in a
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> Hi Dan, Brian
>
> thanks for the comments, i know that for real live samples everything what
> exceeds the resolution limit of the system is an artefact, however for
> sparsely separated beads i would expect that i can determine their position
> with high accuracy only limited by the signal to noise ratio. This is
> exactly what is done in localisation microscopy (PALM, STORM...). One
> approach is to calculate the cross correlation between the measured 3D PSF
> and the 3D image (see e.g. A. G. York et al. Nature Methods 8, 2011, 327).
> While this is not exactly what deconvolution does, it is at least related to
> deconvolution.
>
> So i was wondering if this special case, a limited number of well resolved
> beads which are small enough to qualify as point objects, could tell us how
> good the deconvolution method actually works, or in the case of SIM, what
> the image restauration actually does. At least theoretically, the size of
> the beads (110 nm in my case) should not be a problem as by using the same
> beads to measure the PSF we trick the deconvolution into thinking that they
> are point objects. This is similar to the obove mentioned localisation
> microscopy which does not depend much on optical resolution or pixel size.
>
> best wishes
>
> Andreas
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> -----Original Message-----
> From: Daniel James White <
[hidden email]>
> To:
[hidden email]
> Sent: Mon, 11 Apr 2011 10:53
> Subject: Re: Deconvolution of 3D SIM data
>
>
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> Hi Andreas,
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> On Apr 9, 2011, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system
> wrote:
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> > Date: Fri, 8 Apr 2011 11:48:46 -0400
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> > From: Andreas Bruckbauer <
[hidden email]>
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> > Subject: Re: Deconvolution of 3D SIM data
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> > When i generate a PSF for deconvolution using suitable beads, then image=
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> > the same beads again and deconvolve the image, i would expect to get
> real=
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> > ly tiny dots. See e.g.
http://www.svi.nl/BeadsDeconvolutionExample=20>
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> real tiny? The images of sub resolution objects dont get very much smaller
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> deconvolution...
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> what you really get is much higher contrast, so the features look "sharper"
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> You also get a little bit more resolution.. but thats not really the main
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> Its really about contrast.
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> > I would expect something similar for the OMX when the reconstruction is
> pe=
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> > rfect and includes proper deconvolution. However we get larger features,=
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> > still within the expected OMX resolution 120 nm in width and 300 nm in
> z,=
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> > but no dots.
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> No reconstruction or deconvolution will give images that contain features
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> are smaller
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> than the resolution limit of the system.
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> What do you mean by "dots" ?
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> Single 40 nm pixels?
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> In the OMX it's twice the conventional resolution,
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> so you cant get objects that appear smaller than 120 nm.
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> (actually... API say the OMX system sometimes seems to outperform theory
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> slightly... but only a bit)
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> You should not an must not get single pixel object with the reconstructed
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> spacing of 40 nm (thats the spacing we have with the EM CCDs after 2x
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> increase from the physical 80 nm pixel spacing)
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> If you get smaller objects, its wrong, and an artifact. No?
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> > I think there is still some improvement possible either in=
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> Actually, in my hands it seems to do what theory predicts and no more.
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> There is no good reason to expect it to make smaller objects than 120 nm.
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> Certainly the careful alignment of the optics and the highest quality
> objective
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> lens are critical,
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> as is the measurement of suitable SIM PSFs and careful
> calibration/measurement
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> of the parameters for the reconstruction...
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> the angles and the phases.
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> So, other than making it faster and more tolerant to sub optimal input
> data,
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> I dont think there is any improvement to be made in resolution.. so I dont
> see
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> how you think there is room for resolution improvement?
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> Maybe you can share your ideas?
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> cheers
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> Dan
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> >
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> > best wishes
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> >
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> > Andreas
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> >
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> > =20
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> >
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> > =20
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> Dr. Daniel James White BSc. (Hons.) PhD
>
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>
>
> Senior Microscopist / Image Visualisation, Processing and Analysis
>
>
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>
> Light Microscopy and Image Processing Facilities
>
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>
> Max Planck Institute of Molecular Cell Biology and Genetics
>
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>
> Pfotenhauerstrasse 108
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> 01307 DRESDEN
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> Germany
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> +49 (0)15114966933 (German Mobile)
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> +49 (0)351 210 2627 (Work phone at MPI-CBG)
>
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> +49 (0)351 210 1078 (Fax MPI-CBG LMF)
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>
http://www.bioimagexd.net BioImageXD
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>
http://pacific.mpi-cbg.de Fiji - is just ImageJ (Batteries
> Included)
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>
>
>
http://www.chalkie.org.uk Dan's Homepages
>
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>
>
https://ifn.mpi-cbg.de Dresden Imaging Facility Network
>
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> dan (at) chalkie.org.uk
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> ( white (at) mpi-cbg.de )
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