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Confocal and multiphoton excitation fluorescence deconvolution will 
greatly benefit from customers requiring vendors to include fast, 
quantitative (precise, accurate) deconvolution on all confocal/MPEF 
systems bought/sold. GPU enabled deconvolution. If three academics can 
make GPU SIM (pretty much) realtime (and a heck of a lot faster 
processing than OMX), the hardware vendors ought to be able to do it (or 
license it):

    Lefman J, Scott K, Stranick S. Live, video-rate super-resolution
    microscopy using structured illumination and rapid GPU-based
    parallel processing. Microsc Microanal. 2011 Apr;17(2):191-6. Epub
    2011 Mar 9. PMID: 21385522

If the vendors don't get it done, I hope everyone goes for do it 
yourself nanoscopes, such as:

    *York* AG, Parekh SH, Dalle Nogare D, Fischer RS, Temprine K, Mione
    M, Chitnis AB, Combs CA, *Shroff* H. Resolution doubling in live,
    multicellular organisms via multifocal structured illumination
    microscopy. Nat Methods. 2012 May 13;9(7):749-54. doi:
    10.1038/nmeth.2025. PMID: 22581372
    http://code.google.com/p/msim/

Good luck with the vendors.

***

As for deconvolution "breaking" the optical limit: of course not. The 
deconvolution is done on your computer, the optical part ended when the 
photons slammed into the detector(s). I go with Paul Goodwin's (Applied 
Precision) rule of thumb (or maybe pinky finger?): deconvolution can 
improve the IMAGE resolution by ~20%.

There are other ways to get improvement in resolution - I am very 
pleased with the increase in IMAGE resolution of PiMP:*

*

    *Munck S*, Miskiewicz K, Sannerud R, Menchon SA, Jose L, Heintzmann
    R, Verstreken P, Annaert W. Sub-diffraction imaging on standard
    microscopes through photobleaching microscopy with non-linear
    processing J Cell Sci. 2012 May 1;125(Pt 9):2257-66. PMID: 22357945.

On the Leica SP5 inverted and Zeiss LSM710 confocal microscopes i manage 
here at the U, I use 30 nm pixel size (vs 60 nm for "standard" high 
resolution confocal images), 25 to 50 images (when I'm acquiring for a 
user, my core makes more money if image acquisition time takes longer, 
single channel (makes dealing with Fiji ImageJ easier - would be nice if 
someone took the time to develop ImageJ into a real program with user, 
not anti-user, interface), no averaging, unidirectional scanning. My SP5 
has both standard and resonant scanners - both work (resonant is 
faster). Goal is modest photobleaching (ideally ~5% according to the 
paper). Since 30 nm pixel size is smaller than the PiMP plugin default, 
I use filter size = 1.65. My thanks to Sebastian Munck and Glen 
Macdonald for the recommendation and for putting in 16-bit output mode, 
which I routinely use. Does PiMP work? Yes. Don't take my word for it: 
go look at Figure 2 of the paper and/or get the plugin from the 
corresponding author.

George


On 10/23/2012 9:23 PM, Guy Cox wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There's no simple answer.  Some people have aimed to use deconvolution to go beyond the optical resolution limit.  The best example I know is:
> Carrington, W.A., Lynch, R.M., Moore, E.D.W., Isenberg, G., Fogarty, K.E. and Fay, F.S., 1995.  Superresolution Three-Dimensional Images of Fluorescence in Cells with Minimal Light Exposure. Science 268, 1483-1487
>
> Commercial deconvolution systems are not usually aiming at that.  The aim is to separate in focus and out of focus light and thereby achieve optical sectioning in wide field.  Since the light budget is always better in wide field than in confocal this has certain advantages, but it has the disadvantage that the wide field OTF has a 'missing cone' of directions in which no information is present.  This limits what can be achieved.
>
> The confocal OTF has no missing cone which makes it quite a good target for deconvolution.  The confocal PSF Is far from optimal, being very streaked out in the Z direction, and deconvolution can do a lot to improve it.  Several people (including me) have advocated 1D deconvolution in just the Z direction, which can greatly improve image quality.  My papers on this are a bit hard to find but there is an example in my chapter in Jim Pawley's book.  However deconvolution of confocal images hasn't really caught on as much as it should have.
>
>                                                                                   Guy
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Christophe Leterrier
> Sent: Wednesday, 24 October 2012 3:28 AM
> To: [log in to unmask]
> Subject: Question about deconvolution
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi folks,
>
> I have a long-standing question regarding deconvolution (as processing widefield or confocal images to reassign light from where it originated using a PSF).
>
> Is there a theoretical limit to the resolution one could obtain using deconvolution? Is is theoretically possible to "break" the diffraction limit with deconvolution? That is, to get under the classical 200x200x600nm spot? I think it is not the case, but then why would you deconvolve widefield or confocal images? What do you gain by doing so on a system that is reasonably close to its theoretical capabilities in terms of optical performances?
>
> Thanks for your help,
>
> Christophe
>
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>
>