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Hi Christophe

"Is is theoretically possible to "break" the diffraction limit with
deconvolution? That is, to get under the classical 200x200x600nm
spot?"

A "spot" or point test can be very deceiving.  It is easy to restore a
point like object to a small size under the classical 200x200x600nm
spot.  For example by over-deconvolving with too many iterations or by
using a PSF that has too large an extent.

(As an aside if you have only one (or sparse) point(s)  you often just
want to identify the location which can be done with sub-diffraction
and sub-voxel accuracy).

Another question is can you separate two points a distance of x, y and
z apart when x,y, and z are close to the resolution limit??

When you reassign the light you should get better contrast between two
points if they are resolvable.   Thus a counting routine should do a
better job detecting the 2 points after deconvolution.  But
deconvolution won't separate 2 points with separation smaller than the
limit.   (So Ideally deconvolution algorithms should be evaluated
using at least a 2 point test and not a spot test.)

So to answer your question why deconvolve??  To get better contrast
between structure leading to better measurement of structure (Though
more work could be done on objectively quantifying the benefits of
deconvolution on measurements relevant to biology, especially "barely
resolvable" structure).

Another way to look at it is as follows: If you didn't deconvolve you
would have to integrate more knowledge of the blurring process into
counting and segmentation routines.  It is cleaner to do these steps
separately.

Brian

On Tue, Oct 23, 2012 at 1:08 PM, MODEL, MICHAEL <[log in to unmask]> wrote:
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>
> There are references on the Huygens site about moderate improvement of resolution
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Christophe Leterrier
> Sent: Tuesday, October 23, 2012 12:28 PM
> To: [log in to unmask]
> Subject: Question about deconvolution
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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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