http://confocal-microscopy-list.275.s1.nabble.com/Question-about-deconvolution-tp7579203p7579213.html
Richardson Lucy the FWHM can be smaller than the airy disc. Meaning
structure. For example if the axial FWHM is reduced by 3 (which it
laser ophthalmoscope) emphasized this point. In that case we were
barely resolvable axially. In some cases deconvolution would
signal that actually contained 2 reflectors. So we had to
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> To be honest our group has not done any sort of scientific analysis of the
> resolution improvement you can achieve. On the other hand, we anecdotally
> experienced about 1.5 (possibly 2, but that's pushing it) reduction in FWHM
> along x, y. Generally, along z, our experience has always been that you
> (practically speaking, day-to-day) can't really surpass the depth-of-field.
> On the other hand, with a confocal, you CAN cut that
> axial resolution in about 1/2 (as I recall. It might be a little bit better
> than that actually. Mmmmmm, possibly 3, I don't really remember.). There
> is an interesting paper by Peter Shaw where he takes axial measurements of
> resolution improvement, comparing widefield to confocal, but maybe that's a
> different topic.
>
> The trade-off that comes with the resolution improvement, when you try to go
> past the diffraction limit, is that it comes with ringing (oscillations at
> edges of objects) that gets enhanced in the x,y dimension. That does not
> make the idea useless - you just have to be able to accept the trade-off to
> get the resolving power improvement. There are ways to reduce the ringing
> that we have played with and that actually do work, but to my understanding
> they have not been refined, made to work in a repeatable way and thereby
> commercialized.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]] On
> Behalf Of Lutz Schaefer
> Sent: Tuesday, October 23, 2012 10:16 PM
> To:
[hidden email]
> Subject: Re: Question about deconvolution
>
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>
> I do agree with most of the posts here and like to add a few more things.
>
> From a theoretical point of view and preferably in noiseless continuous
> space, there is a clear limitation of restoration of spatial frequencies up
> to the optical resolution for an unconstrained linear regularized inverse
> system. It will improve the FWHM of a point object, but not beyond the Airy
> disk. Using a nonlinear, for example positivity constrained iterative
> method, this limitation becomes lifted as frequencies beyond that limit are
> allowed to enter (to understand how, there is a paper from Merserau et al.).
>
> Especially in the widefield case, the gain in axial resolution becomes
> easily apparent but only with this class of methods and with proper
> background correction. From the theory, I would see no limitation in
> regaining spatial frequencies beyond optical resolution. Awakening usually
> strikes in the real world, where we deal with photon limited discrete
> measurements as George points out. In the end it is a battle with the signal
> to noise and the ill-posed-ness of the underlying problem to be solved. This
> battle is lost to these factors and regularization has to be chosen to mend
> noise amplification which diminishes the goal as it suppresses high
> frequencies. There are older papers from Jose Conchello and Rainer Heitzmann
> (sorry I don't have the references handy) who try to achieve the utmost
> improvement, but with only mild success. Also Walter Carrington's paper,
> mentioned from Guy lies in this category, but he was rather focusing on a
> very solid rigorous mathematical concept, that would achieve this goal.
>
> To make a long story short. Deconvolution can greatly improve the contrast
> of fine structures. Some methods can in theory break the diffraction limit,
> but with real data this can only be achieved at best in axial dimension of a
> widefield modality.
>
> Regards
> Lutz
>
> __________________________________
> L u t z S c h a e f e r
> Sen. Scientist
> Mathematical modeling / Image processing Advanced Imaging Methodology
> Consultation
> 16-715 Doon Village Rd.
> Kitchener, ON, N2P 2A2, Canada
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> Email:
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>
> --------------------------------------------------
> From: "George McNamara" <
[hidden email]>
> Sent: Tuesday, October 23, 2012 21:48
> To: <
[hidden email]>
> Subject: Re: Question about deconvolution
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> *****
>>
>> 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:
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>>>
>>> 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:
[hidden email]]
>>> On Behalf Of Christophe Leterrier
>>> Sent: Wednesday, 24 October 2012 3:28 AM
>>> To:
[hidden email]
>>> 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
>>>
>>>