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Question about deconvolution

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lechristophe

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

mmodel

Re: Question about deconvolution

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To join, leave or search the confocal microscopy listserv, go to:
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*****

There are references on the Huygens site about moderate improvement of resolution

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Tuesday, October 23, 2012 12:28 PM
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

Julio Vazquez

Re: Question about deconvolution

In reply to this post by lechristophe

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Hi Christophe,

I won't venture into this discussion other than saying that, in widefield microscopy, contast and resolution are interdependent, and that deconvolution improves both by reducing blur (loss of contrast) due to out of focus signal. There are many good reviews and discussions, including a few chapters in the Confocal Handbook. Another good starting point is here:

http://micro.magnet.fsu.edu/primer/digitalimaging/deconvolution/deconvolutionhome.html

One recent interesting example is work from the Smith lab at Stanford where they use deconvolution on thin sections to effectively achieve super-resolution (~ 100 nm). For example, see:

Wang and Smith,
Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens.
PLoS Comput Biol. 2012 Aug;8(8):e1002671. Epub 2012 Aug 30.

Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

http://www.fhcrc.org

==

On Oct 23, 2012, at 9:28 AM, Christophe Leterrier wrote:

> *****
> 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

Brian Northan

Re: Question about deconvolution

In reply to this post by mmodel

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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 <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There are references on the Huygens site about moderate improvement of resolution
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
> Sent: Tuesday, October 23, 2012 12:28 PM
> 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

Guy Cox-2

Re: Question about deconvolution

In reply to this post by lechristophe

*****
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:[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

George McNamara

Re: Question about deconvolution

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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:
> 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:[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
>
>

Lutz Schaefer

Re: Question about deconvolution

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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
Phone/Fax: +1 519 894 8870
Email: [hidden email]
___________________________________

--------------------------------------------------
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:
>> 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:[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
>>
>>

Tim Holmes

Re: Question about deconvolution

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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
Phone/Fax: +1 519 894 8870
Email: [hidden email]
___________________________________

--------------------------------------------------
From: "George McNamara" <[hidden email]>
Sent: Tuesday, October 23, 2012 21:48
To: <[hidden email]>
Subject: Re: Question about deconvolution

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:[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
>>
>>

Brian Northan

Re: Question about deconvolution

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Tim, Lutz

As you both mention "frequencies beyond the limit" are allowed to
enter when using a constrained, iterative and un-regularized method.
For example when deconvolving a point object with unregularized
Richardson Lucy the FWHM can be smaller than the airy disc. Meaning
that frequencies beyond the diffraction limit are present in the
output.

However it is not obvious what the implications are for complicated
structure. For example if the axial FWHM is reduced by 3 (which it
can be) it doesn't mean 2 points with that separation can be resolved.

I remember the work Autoquant/Lickenbrock did with the SLO (scanning
laser ophthalmoscope) emphasized this point. In that case we were
trying to find the location of 2 membranes in the retina that were
barely resolvable axially. In some cases deconvolution would
incorrectly assign one "pulse" with an apparently small FWHM to a
signal that actually contained 2 reflectors. So we had to
investigate other approaches using constrained MLE parameter
estimation.

Brian

On Wed, Oct 24, 2012 at 7:31 AM, Tim Holmes
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> Phone/Fax: +1 519 894 8870
> Email: [hidden email]
> ___________________________________
>
>
> --------------------------------------------------
> 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:
>>> 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:[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
>>>
>>>

Guy Cox-2

Re: Question about deconvolution

In reply to this post by Tim Holmes

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The thing about the confocal axial (Z) resolution is that it has no minimum - it just trails off ad infinitum. This is quite unlike the lateral (XY) resolution, which has a real, defined zero.

This means that the Z resolution looks much worse than it is. A bright object may have (with an NA 1.4 objective) an FWHM of 500nm, but you will still see it at depths 2 or 3 times greater than that. Very simple deconvolution methods can modify this to give an image in which the perceived axial resolution at least looks like the measured one, which is not the case in an unprocessed image, and is of itself a huge practical improvement. As Tim says, we can probably do better that that, but I doubt that we can get it quite isotropic.

G.C. Cox and C. Sheppard, 1993. Effects of image deconvolution on optical sectioning in conventional and confocal microscopes. Bio-Imaging 1, 82-95.

G.C. Cox and Colin Sheppard, 1999 Appropriate Image Processing for Confocal Microscopy. In: P.C. Cheng, P P Hwang, J L. Wu, G Wang & H Kim (eds) Focus on Multidimensional Microscopy. World Scientific Publishing, Singapore, New Jersey, London & Hong Kong. Volume 2, pp 42-54 ISBN 981-02-3992-0

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tim Holmes
Sent: Wednesday, 24 October 2012 10:31 PM
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
*****

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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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
Phone/Fax: +1 519 894 8870
Email: [hidden email]
___________________________________

--------------------------------------------------
From: "George McNamara" <[hidden email]>
Sent: Tuesday, October 23, 2012 21:48
To: <[hidden email]>
Subject: Re: Question about deconvolution

license it):

plugin from the corresponding author.

example I know is:
>> Carrington, W.A., Lynch, R.M., Moore, E.D.W., Isenberg, G., Fogarty, K.E.

mmodel

Re: Question about deconvolution

In reply to this post by Tim Holmes

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If resolution improvement in transmission microscopy were possible, it might be very useful to apply it to interferometry. There you have a nanometer resolution in the vertical dimension, but it all is undermined by diffraction-limited lateral resolution (the situation is the same in "transmission through dye" for cell profile measurements). And since it is not confocal or fluorescence, noise probably would not be that much of an issue. I have talked to a few companies that make interferometric microscopes, it seems that they haven't though much about it.

Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Tim Holmes [[hidden email]]
Sent: Wednesday, October 24, 2012 7:31 AM
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
*****

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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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
Phone/Fax: +1 519 894 8870
Email: [hidden email]
___________________________________

--------------------------------------------------
From: "George McNamara" <[hidden email]>
Sent: Tuesday, October 23, 2012 21:48
To: <[hidden email]>
Subject: Re: Question about deconvolution

license it):

plugin from the corresponding author.

example I know is:
>> Carrington, W.A., Lynch, R.M., Moore, E.D.W., Isenberg, G., Fogarty, K.E.

Guy Cox-2

Re: Question about deconvolution

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

NO! NO! NO!

You do NOT have nanometre RESOLUTION in the axial (vertical, Z) dimension. You have nanometre measurement precision in the axial dimension. You also have nanometre measurement precision in the lateral (XY) dimensions - that is the basis of PALM, STORM etc. Neither property is resolution (the ability to separate two objects).

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: Wednesday, 24 October 2012 11:05 PM
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
*****

If resolution improvement in transmission microscopy were possible, it might be very useful to apply it to interferometry. There you have a nanometer resolution in the vertical dimension, but it all is undermined by diffraction-limited lateral resolution (the situation is the same in "transmission through dye" for cell profile measurements). And since it is not confocal or fluorescence, noise probably would not be that much of an issue. I have talked to a few companies that make interferometric microscopes, it seems that they haven't though much about it.

Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Tim Holmes [[hidden email]]
Sent: Wednesday, October 24, 2012 7:31 AM
To: [hidden email]
Subject: Re: Question about deconvolution

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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
Phone/Fax: +1 519 894 8870
Email: [hidden email]
___________________________________

--------------------------------------------------
From: "George McNamara" <[hidden email]>
Sent: Tuesday, October 23, 2012 21:48
To: <[hidden email]>
Subject: Re: Question about deconvolution

license it):

plugin from the corresponding author.

example I know is:
>> Carrington, W.A., Lynch, R.M., Moore, E.D.W., Isenberg, G., Fogarty, K.E.

Hans

Re: Question about deconvolution

In reply to this post by Guy Cox-2

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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
>
> *****
> 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 ([hidden email])
Scientific Volume Imaging b.v., URL: http://www.svi.nl/

Tim Holmes

Re: Question about deconvolution

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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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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:[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
>
>

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

Hans

Re: Question about deconvolution

*****
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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:[hidden email]] On
> Behalf Of Hans
> Sent: Wednesday, October 24, 2012 7:29 AM
> 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
> *****
>
> 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
>>
>> *****
>> 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 ([hidden email])
> Scientific Volume Imaging b.v., URL: http://www.svi.nl/
>
>

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

mmodel

precision and resolution

In reply to this post by Guy Cox-2

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I believe that the word "resolution" with its low-nm estimates is always applied to STORM, PALM, etc - the ability to tell objects apart.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Wednesday, October 24, 2012 8:16 AM
To: [hidden email]
Subject: Re: Question about deconvolution

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NO! NO! NO!

You do NOT have nanometre RESOLUTION in the axial (vertical, Z) dimension. You have nanometre measurement precision in the axial dimension. You also have nanometre measurement precision in the lateral (XY) dimensions - that is the basis of PALM, STORM etc. Neither property is resolution (the ability to separate two objects).

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: Wednesday, 24 October 2012 11:05 PM
To: [hidden email]
Subject: Re: Question about deconvolution

*****
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If resolution improvement in transmission microscopy were possible, it might be very useful to apply it to interferometry. There you have a nanometer resolution in the vertical dimension, but it all is undermined by diffraction-limited lateral resolution (the situation is the same in "transmission through dye" for cell profile measurements). And since it is not confocal or fluorescence, noise probably would not be that much of an issue. I have talked to a few companies that make interferometric microscopes, it seems that they haven't though much about it.

Mike

Vitaly Boyko

scientific video editing software

*****
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Dear All,

we are looking for an intuitive, user-friendly SCIENTIFIC video editing software, that would also allow, in addition of placing arrows (Image J plugin is available for the purpose), to draw freeform lines (pencil tool), adding/highlighting ROIs, and placing other simple annotation objects.

With best regards,

Vitaly

mskcc
646-888-2186

Kevin Ryan

Re: Question about deconvolution

In reply to this post by lechristophe

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COMMERCIAL RESPONSE - AutoQuant

It's important to note that there are a few different issues here.

The _theoretic_ resolution is driven by the highest observed spatial frequencies, the sharpest edges, that make it through the observing system. This is set by the aperture and wavelength of the observation, an information limit.

The _effective_ resolution, the ability to separate nearby objects, is also dependent on the _strength_ of those frequencies. In a single in focus XY plane (for example), high frequencies drop in strength from 100% at the average value, to zero at the high frequency aperture limit, the sharpest edges. That fall-off blurs the observations considerably.

Deconvolution restores higher frequency strengths (in frequency terms, changing a tapered pyramid centered at zero to a near-box), reducing the full-width half max (FWHM) of observed structures accordingly. You have more edge information, which means sharper edges. Add to that (in widefield) the removal of uncorrelated out of focus illumination, and (particularly in confocal) reduction of noise that's unsupported by a PSF, and you will obtain a considerable improvement in _effective_ resolution, the ability to separate nearby structures.

Deconvolution won't provide superresolution (frequencies past the aperture limit aren't represented in the observation, and in a noise-limited real world situation any estimate would be unstable), but the effective observed deconvolution resolution improvement, separability, in XYZ is ~2-2.5x over the original data.

See Kano 1996 (http://www3.mpibpc.mpg.de/groups/hell/publications/pdf/Bioimaging_4_187-197.pdf - thank you for that reference, Hans) for a discussion of FWHM improvements seen in maximum-likelyhood restoration.

Kevin Ryan
Media Cybernetics, Inc.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Tuesday, October 23, 2012 12:28 PM
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
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Michael Schell-2

Re: Question about deconvolution

In reply to this post by Guy Cox-2

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Another example I've seen divides the pixels in to subpixels and
deconvolves them using Autoquant.

http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=850529

This version is actually included in the Autoquant 3D blind expert
settings. We tested this a few times but gave up because the initial
results did not look any better than when we used the standard Autoquant
blind default settings. The sub-pixel routine took a long time to process
and obviously the output files are much bigger when you make sub-pixels.
We lacked a fast enough computer to allow more systematic testing.
Nevertheless, this is an interesting approach, which further emphasizes
the point made previously that some super-resoluton approaches can be
viewed as a type of deconvolution. A range of super-resolution approaches
(SIM, STED, STORM, PALM, light-sheet) add a deconvolution step post-hoc to
increase signal-to-noise.

Mike

On Tue, Oct 23, 2012 at 9:23 PM, Guy Cox <[hidden email]> 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:[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
>

mahogny

Re: scientific video editing software

In reply to this post by Vitaly Boyko

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

On Wed, Oct 24, 2012 at 5:55 PM, Vitaly Boyko <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> we are looking for an intuitive, user-friendly SCIENTIFIC video editing
> software, that would also allow, in addition of placing arrows (Image J
> plugin is available for the purpose), to draw freeform lines (pencil tool),
> adding/highlighting ROIs, and placing other simple annotation objects.
>

you can have a look at endrov,
www.endrov.net
which places a lot of emphasis on working with large files, and on
annotation. I don't know if all the annotation support you need is in there
yet but if not, let us know how we can improve it

/Johan

>
> With best regards,
>
> Vitaly
>
> mskcc
> 646-888-2186
>

--
--
-----------------------------------------------------------
Johan Henriksson, PhD
Karolinska Institutet
Ecobima AB - Custom solutions for life sciences
http://www.ecobima.com http://mahogny.areta.org http://www.endrov.net

<http://www.endrov.net>