http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-3D-SIM-data-tp6251420p6271259.html
> structure you are labelling. Several studies have done this with great
> effect. Whether this is true super-resolution is something one could
>
> Sponsor my next half-marathon on May 15th
>
> There's a special reason - find it out at
>
>
http://www.everydayhero.com.au/Guy_Cox_4846> <
http://www.everydayhero.com.au/Guy_Cox_4846>
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy & Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>
> Mobile 0413 281 861
>
> ______________________________________________
>
>
http://www.guycox.net <
http://www.guycox.net/>
>
>
>
> From: Confocal Microscopy List [mailto:
[hidden email]]
> On Behalf Of Gustin, Emmanuel [TIBBE]
> Sent: Thursday, 14 April 2011 12:19 AM
> To:
[hidden email]
> Subject: Re: Deconvolution of 3D SIM data
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> I think that actually, the critical point is that you *know* the beads
> (or in the PALM/STORM case, molecules) are sub-resolution in size? The
> image reconstruction uses more information than is present in the image,
> and this allows you to go below the optical resolution limit. But there
> is no way to tell from the image alone what size the beads are.
>
> I guess that theoretically, if you would feed an algorithm with the
> knowledge that you have labeled, for example, tubulin and information
> about the patterns you expect to see (size of structures, persistence
> lengths of bending, branching behavior, ...) you could use it to do
> a type of "deconvolution plus" and create a sub-resolution image.
>
> Whether other people would have confidence in the image that has been
> constructed in that way, is another matter.
>
> Best Regards,
>
> Emmanuel
>
>
> --
> Emmanuel Gustin, Tel. (+32) 14 64 1586, e-mail:
>
[hidden email]
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]]
> On Behalf Of Guy Cox
> Sent: woensdag 13 april 2011 14:34
> To:
[hidden email]
> Subject: Re: Deconvolution of 3D SIM data
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Well, the critical point is the 'sufficiently spaced' bit. If your
> points are well separated you can find their centroids with a precision
> way beyond the Rayleigh diffraction limit, which essentially applies to
> objects separated by the diffraction limit. Rayleigh's criterion is
> only a rule of thumb - with sufficient signal to noise you can separate
> objects closer together than the limit by deconvolution. Abbe's limit,
> on the other hand, is non-negotiable, but it doesn't apply in
> fluorescence. However, when it comes to SIM data, as Nuno has pointed
> out, we have already defined the bandwith we are probing, and there is
> nothing more to get.
>
>
>
>
> Guy
>
>
>
> Sponsor my next half-marathon on May 15th
>
> There's a special reason - find it out at
>
>
http://www.everydayhero.com.au/Guy_Cox_4846> <
http://www.everydayhero.com.au/Guy_Cox_4846>
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy & Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>
> Mobile 0413 281 861
>
> ______________________________________________
>
>
http://www.guycox.net <
http://www.guycox.net/>
>
>
>
> From: Confocal Microscopy List [mailto:
[hidden email]]
> On Behalf Of Christophe Leterrier
> Sent: Wednesday, 13 April 2011 7:31 PM
> To:
[hidden email]
> Subject: Re: Deconvolution of 3D SIM data
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> To elaborate a bit on that, why isn't it possible to go beyond the
> diffraction limit with deconvolution ? I don't see the theoretical
> reason,
> in a ideal case (subresolutive beads sufficiently spaced). Of course in
> a
> real sample, S/N issues and fluorophore density impose a limit on the
> precision, but why would this limit precisely be at the diffraction
> limit ?
>
> Christophe
>
> On Wed, Apr 13, 2011 at 09:44, Andreas Bruckbauer <
[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> *****
>>
>>
>> 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
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> -----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
>>
>>
>> *****
>>
>>
>>
>>
>>
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>
>>
>>
>>
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>
>>
>>
>>
>>
>> *****
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Hi Andreas,
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> On Apr 9, 2011, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system
>> wrote:
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> > Date: Fri, 8 Apr 2011 11:48:46 -0400
>>
>>
>>
>>
>>
>> > From: Andreas Bruckbauer <
[hidden email]>
>>
>>
>>
>>
>>
>> > Subject: Re: Deconvolution of 3D SIM data
>>
>>
>>
>>
>>
>> >
>>
>>
>>
>>
>>
>> >
>>
>>
>>
>>
>>
>> > When i generate a PSF for deconvolution using suitable beads, then
> image=
>>
>>
>>
>>
>>
>> > the same beads again and deconvolve the image, i would expect to get
>> real=
>>
>>
>>
>>
>>
>> > ly tiny dots. See e.g.
>
http://www.svi.nl/BeadsDeconvolutionExample=20>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> real tiny? The images of sub resolution objects dont get very much
> smaller
>> after
>>
>>
>>
>>
>>
>> deconvolution...
>>
>>
>>
>>
>>
>> what you really get is much higher contrast, so the features look
> "sharper"
>>
>>
>>
>>
>>
>> You also get a little bit more resolution.. but thats not really the
> main
>> point.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Its really about contrast.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> > I would expect something similar for the OMX when the reconstruction
> is
>> pe=
>>
>>
>>
>>
>>
>> > rfect and includes proper deconvolution. However we get larger
> features,=
>>
>>
>>
>>
>>
>> > still within the expected OMX resolution 120 nm in width and 300 nm
> in
>> z,=
>>
>>
>>
>>
>>
>> > but no dots.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> No reconstruction or deconvolution will give images that contain
> features
>> that
>>
>>
>>
>>
>>
>> are smaller
>>
>>
>>
>>
>>
>> than the resolution limit of the system.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> What do you mean by "dots" ?
>>
>>
>>
>>
>>
>> Single 40 nm pixels?
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> In the OMX it's twice the conventional resolution,
>>
>>
>>
>>
>>
>> so you cant get objects that appear smaller than 120 nm.
>>
>>
>>
>>
>>
>> (actually... API say the OMX system sometimes seems to outperform
> theory
>>
>>
>>
>>
>>
>> slightly... but only a bit)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> You should not an must not get single pixel object with the
> reconstructed
>> pixel
>>
>>
>>
>>
>>
>> spacing of 40 nm (thats the spacing we have with the EM CCDs after 2x
>> resolution
>>
>>
>>
>>
>>
>> increase from the physical 80 nm pixel spacing)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> If you get smaller objects, its wrong, and an artifact. No?
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> > I think there is still some improvement possible either in=
>>
>>
>>
>>
>>
>> > the setup of the instrument or the software.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Actually, in my hands it seems to do what theory predicts and no more.
>>
>>
>>
>>
>>
>> There is no good reason to expect it to make smaller objects than 120
> nm.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Certainly the careful alignment of the optics and the highest quality
>> objective
>>
>>
>>
>>
>>
>> lens are critical,
>>
>>
>>
>>
>>
>> as is the measurement of suitable SIM PSFs and careful
>> calibration/measurement
>>
>>
>>
>>
>>
>> of the parameters for the reconstruction...
>>
>>
>>
>>
>>
>> the angles and the phases.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> So, other than making it faster and more tolerant to sub optimal input
>> data,
>>
>>
>>
>>
>>
>> I dont think there is any improvement to be made in resolution.. so I
> dont
>> see
>>
>>
>>
>>
>>
>> how you think there is room for resolution improvement?
>>
>>
>>
>>
>>
>> Maybe you can share your ideas?
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> cheers
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Dan
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> >
>>
>>
>>
>>
>>
>> > best wishes
>>
>>
>>
>>
>>
>> >
>>
>>
>>
>>
>>
>> > Andreas
>>
>>
>>
>>
>>
>> >
>>
>>
>>
>>
>>
>> >
>>
>>
>>
>>
>>
>> > =20
>>
>>
>>
>>
>>
>> >
>>
>>
>>
>>
>>
>> > =20
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> 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 )
>>
>>
>>
>>
>>
>>
>>
>>
>>
>
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