*****
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*****
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: [log in to unmask]
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Guy Cox
Sent: woensdag 13 april 2011 14:34
To: [log in to unmask]
Subject: Re: Deconvolution of 3D SIM data
*****
To join, leave or search the confocal microscopy listserv, go to:
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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:[log in to unmask]]
On Behalf Of Christophe Leterrier
Sent: Wednesday, 13 April 2011 7:31 PM
To: [log in to unmask]
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 <[log in to unmask]>
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 <[log in to unmask]>
> To: [log in to unmask]
> 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 <[log in to unmask]>
>
>
>
>
>
> > 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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