Posted by
Tim Holmes on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Question-about-deconvolution-tp7579203p7579217.html
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All good points, Hans. A point to keep in mind is that the magnitude of the
numbers can be deceiving.
A 50% improvement in resolving power, for instance, sound BORING to someone
who is just hearing the numbers.
But if you look at images side-by-side, you can make out much more detail in
the one with 50% better resolving power (in my opinion; you be the judge).
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On
Behalf Of Hans
Sent: Wednesday, October 24, 2012 7:29 AM
To:
[hidden email]
Subject: Re: Question about deconvolution
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Dear All,
<message from a commercial vendor>
> 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.
>
In addition to Lutz' remarks, one could say that the widefield missing cone
represents a severe limit on certain frequencies of the object, and the
limit is imposed by diffraction. Depending on the sparseness of the object,
and exploiting extra a priori knowledge besides knowledge of the PSF like
non-negativity, noise characteristics (Poisson noise), possibly properties
of the object itself, it is possible te reconstruct the missing frequencies
-- but not always. Reconstruction of frequencies outside the bounding box of
the widefield OTF is also possible, but requires good optical conditions and
low noise to start with.
To give an extreme example: one can consider localization estimation in
STORM as a special deconvolution case, one where a very powerful bit of
a-priori knowledge is used: there is just one emitter present. The
attainable accuracy then remains controlled by noise and the width of the
PSF, but is much better than the width of the PSF. Deconvolution with
standard methods of widefield images of point objects is also usually easy.
> 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.
In the confocal case the theoretical bandwidth is a good deal beyond the
effective, noise determined limit. So there it is very hard to go beyond the
theoretical limit, but improving on the effective bandlimit is nearly always
possible, especially in Z. In my experience, it is often possible to gain 2x
in Z, somewhat less, say 50%, in XY. This means that you go into the
direction of isotropic resolution, but will not reach it. In good optical
condition the measurement volume may go down by a factor 8.
Lastly, systems like STED which are based on a strong non-linear effect do
not have a hard diffraction imposed bandlimit anymore. They still have an
effective bandlimit, which we find deconvolution can improve much on.
-- Hans
SVI-Huygens
> -----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
>
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>
> 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
>
>
--
-------------------------------------------------------------------
dr. Hans T.M. van der Voort (
[hidden email])
Scientific Volume Imaging b.v., URL:
http://www.svi.nl/