Posted by
Daniel James White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-3D-SIM-data-tp6251420p6252864.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Hi Amro and David,
> Date: Thu, 7 Apr 2011 13:43:45 -0700
> From: David Baddeley <
[hidden email]>
> Subject: Re: Deconvolution of 3D SIM data
>
>
>> Date: Thu, 7 Apr 2011 15:16:08 -0500
>> From: Amro Hamdoun <
[hidden email]>
>> Subject: Deconvolution of 3D SIM data
>>
>>
>> Hi Everyone,
>>
>> We are starting to process through OMX data sets and we are writing for =
>> any=20
>> thoughts on the use of deconvolution in these images.
We got our OMX installed early this year..... this is what I have learned so far.....
in the Applied precision softworx software, the tool that does the SIM reconstruction
takes a measured SIM PSF and uses that to deconvolve the images,
as part of the super resolution reconstruction.
Whichever engineer or application specialist from API that installed your OMX
should have trained at least a couple of folks at your site on all this stuff.
Maybe that was not you.
Anyone who uses this kind of complex system needs to understand its optics
and the ideas behind the maths that do the reconstruction ( = modeling of the fluorophore distribution)
be it standard widefield deconvolution (eg DeltaVision), normal structured illumination (apotome / optiogrid) , super resolution structured illumination (OMX), PALM/STORM ... etc.
Without that understanding, you have no hope of sensibly interpreting the output images,
and deciding if the image contains bad artifacts or not.
Applied Precision run advanced user training sessions,
and if you want to learn more about what is going on, you might like to arrange to go to Seattle and take part in one.
Failing that, you should read the original papers by Matts Gustaffson that describe the methods.
http://www.hhmi.org/research/groupleaders/gustafsson_bio.html>> We have heard mixed=
>> =20
>> things about whether or not it is valid... Has anyone here deconvolved 3=
>> D SIM=20
>> data? Were actual or theoretical PSFs used?=20
the OMX software provided by API for 3D SIM super resolution reconstruction,
does deconvolution with a measured SIM PSF (not the same as the wide field one)
as part of the image reconstruction.
thus it makes no sense to do an extra deconvoution afterwards...
the result images ia already deconvolved: its a most likely model of the fluorophore distribution,
resolution limited (spatial frequency band limited) by the physics of the system.
>>
>> Much thanks,
>> A
>
> I believe that SIM data is usually at least partly deconvolved already in t=
> he =0Areconstruction process, although the extent of the deconvolution depe=
> nds on the =0Aheritage of your SIM technique.
OMX software does a full deconvolution, as far as I understand.
> I think all techniques involv=
> e at least a Weiner =0Afiltering step, and some might include a iterative o=
> ptimisation as well. I'd =0Athus be pretty cautious about performing any fu=
> rther deconvolution.
Me too.
> If you do, =0Ayou'd want to use very small beads (<50 =
> nm) for your PSF measurements, which =0Amight make getting reliable (ie not=
> noisy) PSF estimates difficult.
The OMX software needs measured SIM PSF image (one per camera/channel) for the reconstruction...
the engineer that installed it should have made these,
and one should probably make them again every now and then to test system performance.
> Its also =0Aquite likely that the factors =
> which end up limiting the resolution will relate =0Ato uneven bleaching ('b=
> urn in' of the mask) and mask errors (alignment errors =0Aand sample abbera=
> tions which change the shape of the illumination pattern from =0Awhat is ex=
> pected). Deconvolution isn't going to help you here.=0A=0Acheers,=0ADavid=
David is one the money here...
but you really should talk to someone like Paul Goodwin
or one of the application specialists for OMX at at API.
cheers
Dan
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 )