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Hi Tim


> 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).

Indeed, 50% doesn't sound much..., though for example in camera-land 
manufacturers are proud to gain a mere 50% resolution, but they'll talk 
about doubling the pixel count.

In the paper below measurements were done on two-photon microscope 
images of beads, resulting in a axial gain of 3.5 and a lateral gain of 
2.8. The optical conditions were very well controlled, and beads are 
easy, but these numbers could serve as a practical limit of what one can 
expect.

Kano, Hiroshi, Hans T.M. van der Voort, Martin Schrader, Geert M.P. van 
Kempen and Stefan W. Hell. (1996) Avalanche photodiode detection with 
object scanning and image restoration provided 2-4 fold resolution 
increase in two-photon fluorescence microscopy. BioImaging 4 '96 p187-197.

-- Hans

>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On
> Behalf Of Hans
> Sent: Wednesday, October 24, 2012 7:29 AM
> To: [log in to unmask]
> 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
> *****
>
> 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:[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
>>
>>
>
>
> --
> -------------------------------------------------------------------
> dr. Hans T.M. van der Voort                           ([log in to unmask])
> Scientific Volume Imaging b.v.,             URL: http://www.svi.nl/
>
>


-- 
-------------------------------------------------------------------
dr. Hans T.M. van der Voort                           ([log in to unmask])
Scientific Volume Imaging b.v.,             URL: http://www.svi.nl/