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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
-----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
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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
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http://www.chalkie.org.uk Dan's Homepages
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dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )
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