Deconvolution of Confocal Images? (was: Airy Units)

[Daniel James White]

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Hi Peter,

On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system wrote:

>
> Date:    Sun, 30 Oct 2011 13:09:10 -0700
> From:    Peter Werner <[hidden email]>
> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>
> An interesting point was made here by Jim Pawley:
>
>> I agree that sampling a bit higher than Nyquist never hurts,  
>> especially if you deconvolve (as you always should), but I think  
>> that it is a mistake to think that one can "separate" out the noise  
>> by decon. I think that noise is pretty fundamental.
>
> I had always heard that if you're doing confocal microscopy, at least  
> point-scanning confocal with a pinhole size of 1AU or smaller, that  
> deconvolution was superfluous, because you shouldn't be getting out of  
> focus light. So what is gained by deconvolution when one is sampling  
> voxel by voxel?

in a confocal you throw away most of the signal, as its out of focus.
So as a result the images are often very noisy.
Good contrast.... but high Poisson distributed photon shot noise
from only measuring a handful of photons.

So usually one needs to do something about that noise...
we want to separate the real signal from the noise.

Often a Gaussian or mean filter is applied... which suppresses the noise
by smoothing it out... but it also smooths the real signal, so effectively you lose
the contrast and resolution that was the whole point of doing confocal.

The smart way to suppress the noise, but keep the contrast and resolution
is to do deconvolution.
Deconvolution using a max likelyhood method uses the known shape of the PSF
to make a best guess model of the real fluorophore distribution in the sample.
You tell the deconvolution algorithm how noisy the image is (you have to guess
unless you take 2 images and measure it)
then it attempts to throw out the noise and keep the real signal,
resolution and contrast intact.

D

>
> Peter G. Werner
> Merritt College Microscopy Program

Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666 Skype
http://www.bioimagexd.net  BioImageXD
http://fiji.sc                                        Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Biopolis Dresden Imaging Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Brian Northan]

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Dan

You actually don't have to tell a "pure" MLE deconvolution how noisy
it is... this information is actually embedded in the PSF itself.
When the PSF extends over many pixels, the algorithm can then
differentiate between structure and noise.

If the PSF extends over many pixels then real sub-resolution features
will also extend over many pixels.  Within the MLE update equation a
correlation is done between the PSF and  the image.  PSF and real
features have high correlation but PSF and noise do not.

Many deconvolution implementations have extra noise reduction routines
embedded into the algorithm as constraints.  You ussually need to give
these algorithms an estimate of the noise.  (And there are also some
variations on classic MLE that handle noise in a more explicit
manner).

Thus if the decon needs a noise estimate there is either a separate
noise reduction routine integrated into it, or it is a extension on
classic MLE.

Brian


On Mon, Oct 31, 2011 at 5:29 AM, daniel white <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Peter,
>
> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system wrote:
>
>>
>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>> From:    Peter Werner <[hidden email]>
>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> An interesting point was made here by Jim Pawley:
>>
>>> I agree that sampling a bit higher than Nyquist never hurts,
>>> especially if you deconvolve (as you always should), but I think
>>> that it is a mistake to think that one can "separate" out the noise
>>> by decon. I think that noise is pretty fundamental.
>>
>> I had always heard that if you're doing confocal microscopy, at least
>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>> deconvolution was superfluous, because you shouldn't be getting out of
>> focus light. So what is gained by deconvolution when one is sampling
>> voxel by voxel?
>
> in a confocal you throw away most of the signal, as its out of focus.
> So as a result the images are often very noisy.
> Good contrast.... but high Poisson distributed photon shot noise
> from only measuring a handful of photons.
>
> So usually one needs to do something about that noise...
> we want to separate the real signal from the noise.
>
> Often a Gaussian or mean filter is applied... which suppresses the noise
> by smoothing it out... but it also smooths the real signal, so effectively you lose
> the contrast and resolution that was the whole point of doing confocal.
>
> The smart way to suppress the noise, but keep the contrast and resolution
> is to do deconvolution.
> Deconvolution using a max likelyhood method uses the known shape of the PSF
> to make a best guess model of the real fluorophore distribution in the sample.
> You tell the deconvolution algorithm how noisy the image is (you have to guess
> unless you take 2 images and measure it)
> then it attempts to throw out the noise and keep the real signal,
> resolution and contrast intact.
>
> D
>
>>
>> Peter G. Werner
>> Merritt College Microscopy Program
>
> Dr. Daniel James White BSc. (Hons.) PhD
>
> Leader - Image Processing Facility,
> Senior Microscopist,
> Light Microscopy Facility.
>
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
> [phone] (Work phone at MPI-CBG)
> [phone] (Fax MPI-CBG LMF)
> chalkie666                                      Skype
> http://www.bioimagexd.net       BioImageXD
> http://fiji.sc                                  Fiji -  is just ImageJ (Batteries Included)
> http://www.chalkie.org.uk               Dan's Homepages
> https://ifn.mpi-cbg.de                  Biopolis Dresden Imaging Platform (BioDIP)
> dan (at) chalkie.org.uk
> ( white (at) mpi-cbg.de )
>

[Guy Cox-2]

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Daniel White wrote:

" in a confocal you throw away most of the signal, as its out of focus.
So as a result the images are often very noisy.  "

This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
noise, not signal.  If you have no SA (and, honestly, if you are
seriously interested in high-resolution imaging that should be a given)
then a confocal microscope with the pinhole set at 1 Airy diameter
throws away no signal at all.  So why are confocal images often noisy?
Well, it's just statistics.  If you take a wide-field image with a 1
second exposure each point is exposed for one second.  If you take a
confocal image at 512 x 512 for 1 second then each point is exposed for
~4 microseconds.  The difference is rather substantial ...

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of daniel white
Sent: Monday, 31 October 2011 8:30 PM
To: [hidden email]
Subject: Deconvolution of Confocal Images? (was: Airy Units)

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

Hi Peter,

On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
wrote:

>
> Date:    Sun, 30 Oct 2011 13:09:10 -0700
> From:    Peter Werner <[hidden email]>
> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>
> An interesting point was made here by Jim Pawley:
>
>> I agree that sampling a bit higher than Nyquist never hurts,  
>> especially if you deconvolve (as you always should), but I think  
>> that it is a mistake to think that one can "separate" out the noise  
>> by decon. I think that noise is pretty fundamental.
>
> I had always heard that if you're doing confocal microscopy, at least

> point-scanning confocal with a pinhole size of 1AU or smaller, that  
> deconvolution was superfluous, because you shouldn't be getting out of

> focus light. So what is gained by deconvolution when one is sampling  
> voxel by voxel?

in a confocal you throw away most of the signal, as its out of focus.
So as a result the images are often very noisy.
Good contrast.... but high Poisson distributed photon shot noise
from only measuring a handful of photons.

So usually one needs to do something about that noise...
we want to separate the real signal from the noise.

Often a Gaussian or mean filter is applied... which suppresses the noise

by smoothing it out... but it also smooths the real signal, so
effectively you lose
the contrast and resolution that was the whole point of doing confocal.

The smart way to suppress the noise, but keep the contrast and
resolution
is to do deconvolution.
Deconvolution using a max likelyhood method uses the known shape of the
PSF
to make a best guess model of the real fluorophore distribution in the
sample.
You tell the deconvolution algorithm how noisy the image is (you have to
guess
unless you take 2 images and measure it)
then it attempts to throw out the noise and keep the real signal,
resolution and contrast intact.

D

>
> Peter G. Werner
> Merritt College Microscopy Program

Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666 Skype
http://www.bioimagexd.net  BioImageXD
http://fiji.sc                                        Fiji -  is just ImageJ
(Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Biopolis Dresden Imaging
Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

-----
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Checked by AVG - www.avg.com
Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11

[Jeremy Adler-4]

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

The photons that are rejected by a pinhole usually come from  
fluorophores within the specimen - deconvolution can be viewed as an  
attempt to improve images by putting photons back to where they  
probably originated, rather than to just reject them. Deconvolution  
makes a more efficient use of the emitted photons.

It is therefore possible to obtain an image by deconvolving a  
widefield z series that, because of photobleaching and rejection of  
photons by a pinhole, cannot be obtained from a confocal, even if  
acquisition time was not the limiting constraint.

Only in the narrowest sense, when only a single optical section is  
required, is Guy correct in regarding out of focus fluorescence as  
noise - perhaps signal of unwanted origin.






Quoting Guy Cox <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Daniel White wrote:
>
> " in a confocal you throw away most of the signal, as its out of focus.
> So as a result the images are often very noisy.  "
>
> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
> noise, not signal.  If you have no SA (and, honestly, if you are
> seriously interested in high-resolution imaging that should be a given)
> then a confocal microscope with the pinhole set at 1 Airy diameter
> throws away no signal at all.  So why are confocal images often noisy?
> Well, it's just statistics.  If you take a wide-field image with a 1
> second exposure each point is exposed for one second.  If you take a
> confocal image at 512 x 512 for 1 second then each point is exposed for
> ~4 microseconds.  The difference is rather substantial ...
>
>                                            Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of daniel white
> Sent: Monday, 31 October 2011 8:30 PM
> To: [hidden email]
> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Peter,
>
> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
> wrote:
>
>>
>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>> From:    Peter Werner <[hidden email]>
>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> An interesting point was made here by Jim Pawley:
>>
>>> I agree that sampling a bit higher than Nyquist never hurts,
>>> especially if you deconvolve (as you always should), but I think
>>> that it is a mistake to think that one can "separate" out the noise
>>> by decon. I think that noise is pretty fundamental.
>>
>> I had always heard that if you're doing confocal microscopy, at least
>
>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>> deconvolution was superfluous, because you shouldn't be getting out of
>
>> focus light. So what is gained by deconvolution when one is sampling
>> voxel by voxel?
>
> in a confocal you throw away most of the signal, as its out of focus.
> So as a result the images are often very noisy.
> Good contrast.... but high Poisson distributed photon shot noise
> from only measuring a handful of photons.
>
> So usually one needs to do something about that noise...
> we want to separate the real signal from the noise.
>
> Often a Gaussian or mean filter is applied... which suppresses the noise
>
> by smoothing it out... but it also smooths the real signal, so
> effectively you lose
> the contrast and resolution that was the whole point of doing confocal.
>
> The smart way to suppress the noise, but keep the contrast and
> resolution
> is to do deconvolution.
> Deconvolution using a max likelyhood method uses the known shape of the
> PSF
> to make a best guess model of the real fluorophore distribution in the
> sample.
> You tell the deconvolution algorithm how noisy the image is (you have to
> guess
> unless you take 2 images and measure it)
> then it attempts to throw out the noise and keep the real signal,
> resolution and contrast intact.
>
> D
>
>>
>> Peter G. Werner
>> Merritt College Microscopy Program
>
> Dr. Daniel James White BSc. (Hons.) PhD
>
> Leader - Image Processing Facility,
> Senior Microscopist,
> Light Microscopy Facility.
>
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
> [phone] (Work phone at MPI-CBG)
> [phone] (Fax MPI-CBG LMF)
> chalkie666 Skype
> http://www.bioimagexd.net  BioImageXD
> http://fiji.sc                                        Fiji -  is just ImageJ
> (Batteries Included)
> http://www.chalkie.org.uk                Dan's Homepages
> https://ifn.mpi-cbg.de  Biopolis Dresden Imaging
> Platform (BioDIP)
> dan (at) chalkie.org.uk
> ( white (at) mpi-cbg.de )
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[Guy Cox-2]

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

" The photons that are rejected by a pinhole usually come from  
fluorophores within the specimen - deconvolution can be viewed as an  
attempt to improve images by putting photons back to where they  
probably originated, rather than to just reject them. Deconvolution  
makes a more efficient use of the emitted photons.

It is therefore possible to obtain an image by deconvolving a  
widefield z series that, because of photobleaching and rejection of  
photons by a pinhole, cannot be obtained from a confocal, even if  
acquisition time was not the limiting constraint.

Only in the narrowest sense, when only a single optical section is  
required, is Guy correct in regarding out of focus fluorescence as  
noise - perhaps signal of unwanted origin."

WRONG!!  Yes, of course the photons come from fluorophores within the specimen.  Where else could they come from?  But they don't come from where we are looking at and so they cannot be assigned to the plane we are imaging.  They belong in a different plane and should be assigned there.  And in a confocal stack that is exactly what will happen.  So of course there is wasted fluorescence - and if we want to avoid this the answer is rather simple - use 2-photon. Then there is NO out of plane fluorescence.  

                                     Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
Sent: Monday, 31 October 2011 11:08 PM
To: [hidden email]
Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)

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




Quoting Guy Cox <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Daniel White wrote:
>
> " in a confocal you throw away most of the signal, as its out of focus.
> So as a result the images are often very noisy.  "
>
> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
> noise, not signal.  If you have no SA (and, honestly, if you are
> seriously interested in high-resolution imaging that should be a given)
> then a confocal microscope with the pinhole set at 1 Airy diameter
> throws away no signal at all.  So why are confocal images often noisy?
> Well, it's just statistics.  If you take a wide-field image with a 1
> second exposure each point is exposed for one second.  If you take a
> confocal image at 512 x 512 for 1 second then each point is exposed for
> ~4 microseconds.  The difference is rather substantial ...
>
>                                            Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of daniel white
> Sent: Monday, 31 October 2011 8:30 PM
> To: [hidden email]
> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Peter,
>
> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
> wrote:
>
>>
>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>> From:    Peter Werner <[hidden email]>
>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> An interesting point was made here by Jim Pawley:
>>
>>> I agree that sampling a bit higher than Nyquist never hurts,
>>> especially if you deconvolve (as you always should), but I think
>>> that it is a mistake to think that one can "separate" out the noise
>>> by decon. I think that noise is pretty fundamental.
>>
>> I had always heard that if you're doing confocal microscopy, at least
>
>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>> deconvolution was superfluous, because you shouldn't be getting out of
>
>> focus light. So what is gained by deconvolution when one is sampling
>> voxel by voxel?
>
> in a confocal you throw away most of the signal, as its out of focus.
> So as a result the images are often very noisy.
> Good contrast.... but high Poisson distributed photon shot noise
> from only measuring a handful of photons.
>
> So usually one needs to do something about that noise...
> we want to separate the real signal from the noise.
>
> Often a Gaussian or mean filter is applied... which suppresses the noise
>
> by smoothing it out... but it also smooths the real signal, so
> effectively you lose
> the contrast and resolution that was the whole point of doing confocal.
>
> The smart way to suppress the noise, but keep the contrast and
> resolution
> is to do deconvolution.
> Deconvolution using a max likelyhood method uses the known shape of the
> PSF
> to make a best guess model of the real fluorophore distribution in the
> sample.
> You tell the deconvolution algorithm how noisy the image is (you have to
> guess
> unless you take 2 images and measure it)
> then it attempts to throw out the noise and keep the real signal,
> resolution and contrast intact.
>
> D
>
>>
>> Peter G. Werner
>> Merritt College Microscopy Program
>
> Dr. Daniel James White BSc. (Hons.) PhD
>
> Leader - Image Processing Facility,
> Senior Microscopist,
> Light Microscopy Facility.
>
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
> [phone] (Work phone at MPI-CBG)
> [phone] (Fax MPI-CBG LMF)
> chalkie666 Skype
> http://www.bioimagexd.net  BioImageXD
> http://fiji.sc                                        Fiji -  is just ImageJ
> (Batteries Included)
> http://www.chalkie.org.uk                Dan's Homepages
> https://ifn.mpi-cbg.de  Biopolis Dresden Imaging
> Platform (BioDIP)
> dan (at) chalkie.org.uk
> ( white (at) mpi-cbg.de )
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11

[Mark Cannell]

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

Quite so. If you want to see the effect of deconvolution of 2p images on noise, look here:

Soeller, C. & Cannell, M.B. (1999) Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques. Circ. Res. 84: 266-275

Shameless plug I know, but probably among the first to demonstrate the benefit of decon. to control Poisson noise in confocal/2P...

Mark


On 31/10/2011, at 12:47 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> " The photons that are rejected by a pinhole usually come from  
> fluorophores within the specimen - deconvolution can be viewed as an  
> attempt to improve images by putting photons back to where they  
> probably originated, rather than to just reject them. Deconvolution  
> makes a more efficient use of the emitted photons.
>
> It is therefore possible to obtain an image by deconvolving a  
> widefield z series that, because of photobleaching and rejection of  
> photons by a pinhole, cannot be obtained from a confocal, even if  
> acquisition time was not the limiting constraint.
>
> Only in the narrowest sense, when only a single optical section is  
> required, is Guy correct in regarding out of focus fluorescence as  
> noise - perhaps signal of unwanted origin."
>
> WRONG!!  Yes, of course the photons come from fluorophores within the specimen.  Where else could they come from?  But they don't come from where we are looking at and so they cannot be assigned to the plane we are imaging.  They belong in a different plane and should be assigned there.  And in a confocal stack that is exactly what will happen.  So of course there is wasted fluorescence - and if we want to avoid this the answer is rather simple - use 2-photon. Then there is NO out of plane fluorescence.  
>
>                                     Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
> Sent: Monday, 31 October 2011 11:08 PM
> To: [hidden email]
> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
>
>
> Quoting Guy Cox <[hidden email]>:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Daniel White wrote:
>>
>> " in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.  "
>>
>> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>> noise, not signal.  If you have no SA (and, honestly, if you are
>> seriously interested in high-resolution imaging that should be a given)
>> then a confocal microscope with the pinhole set at 1 Airy diameter
>> throws away no signal at all.  So why are confocal images often noisy?
>> Well, it's just statistics.  If you take a wide-field image with a 1
>> second exposure each point is exposed for one second.  If you take a
>> confocal image at 512 x 512 for 1 second then each point is exposed for
>> ~4 microseconds.  The difference is rather substantial ...
>>
>>                                           Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>     http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of daniel white
>> Sent: Monday, 31 October 2011 8:30 PM
>> To: [hidden email]
>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Peter,
>>
>> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>> wrote:
>>
>>>
>>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>> From:    Peter Werner <[hidden email]>
>>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>> An interesting point was made here by Jim Pawley:
>>>
>>>> I agree that sampling a bit higher than Nyquist never hurts,
>>>> especially if you deconvolve (as you always should), but I think
>>>> that it is a mistake to think that one can "separate" out the noise
>>>> by decon. I think that noise is pretty fundamental.
>>>
>>> I had always heard that if you're doing confocal microscopy, at least
>>
>>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>>> deconvolution was superfluous, because you shouldn't be getting out of
>>
>>> focus light. So what is gained by deconvolution when one is sampling
>>> voxel by voxel?
>>
>> in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.
>> Good contrast.... but high Poisson distributed photon shot noise
>> from only measuring a handful of photons.
>>
>> So usually one needs to do something about that noise...
>> we want to separate the real signal from the noise.
>>
>> Often a Gaussian or mean filter is applied... which suppresses the noise
>>
>> by smoothing it out... but it also smooths the real signal, so
>> effectively you lose
>> the contrast and resolution that was the whole point of doing confocal.
>>
>> The smart way to suppress the noise, but keep the contrast and
>> resolution
>> is to do deconvolution.
>> Deconvolution using a max likelyhood method uses the known shape of the
>> PSF
>> to make a best guess model of the real fluorophore distribution in the
>> sample.
>> You tell the deconvolution algorithm how noisy the image is (you have to
>> guess
>> unless you take 2 images and measure it)
>> then it attempts to throw out the noise and keep the real signal,
>> resolution and contrast intact.
>>
>> D
>>
>>>
>>> Peter G. Werner
>>> Merritt College Microscopy Program
>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>>
>> Leader - Image Processing Facility,
>> Senior Microscopist,
>> Light Microscopy Facility.
>>
>> Max Planck Institute of Molecular Cell Biology and Genetics
>> Pfotenhauerstrasse 108
>> 01307 DRESDEN
>> Germany
>>
>> [phone] (German Mobile)
>> [phone] (Work phone at MPI-CBG)
>> [phone] (Fax MPI-CBG LMF)
>> chalkie666 Skype
>> http://www.bioimagexd.net  BioImageXD
>> http://fiji.sc                                        Fiji -  is just ImageJ
>> (Batteries Included)
>> http://www.chalkie.org.uk                Dan's Homepages
>> https://ifn.mpi-cbg.de  Biopolis Dresden Imaging
>> Platform (BioDIP)
>> dan (at) chalkie.org.uk
>> ( white (at) mpi-cbg.de )
>>
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
>> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>
>
>
>
> Jeremy Adler
> IGP
> Rudbeckslaboratoriet
> Daghammersköljdsväg 20
> 751 85 Uppsala
> Sweden
>
> 0046 (0)18 471 4607
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11

[Brian Northan]

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

The situation is complex because it is highly dependent on sample structure.

In my experience Jeremy is right that in certain cases (sparse sample
structure) deconvolution + widefield produces very nice results
because of out of focus photon reallocation.

But it only works for certain sample structure because of the "missing cone".

There are nice presentations on this (if I recall correctly I saw it
at the woods hole course) comparing widefield + deconvolution and
confocal results for different sample structure.

Brian

On Mon, Oct 31, 2011 at 9:03 AM, Mark Cannell <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Quite so. If you want to see the effect of deconvolution of 2p images on noise, look here:
>
> Soeller, C. & Cannell, M.B. (1999) Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques. Circ. Res. 84: 266-275
>
> Shameless plug I know, but probably among the first to demonstrate the benefit of decon. to control Poisson noise in confocal/2P...
>
> Mark
>
>
> On 31/10/2011, at 12:47 PM, Guy Cox wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> " The photons that are rejected by a pinhole usually come from
>> fluorophores within the specimen - deconvolution can be viewed as an
>> attempt to improve images by putting photons back to where they
>> probably originated, rather than to just reject them. Deconvolution
>> makes a more efficient use of the emitted photons.
>>
>> It is therefore possible to obtain an image by deconvolving a
>> widefield z series that, because of photobleaching and rejection of
>> photons by a pinhole, cannot be obtained from a confocal, even if
>> acquisition time was not the limiting constraint.
>>
>> Only in the narrowest sense, when only a single optical section is
>> required, is Guy correct in regarding out of focus fluorescence as
>> noise - perhaps signal of unwanted origin."
>>
>> WRONG!!  Yes, of course the photons come from fluorophores within the specimen.  Where else could they come from?  But they don't come from where we are looking at and so they cannot be assigned to the plane we are imaging.  They belong in a different plane and should be assigned there.  And in a confocal stack that is exactly what will happen.  So of course there is wasted fluorescence - and if we want to avoid this the answer is rather simple - use 2-photon. Then there is NO out of plane fluorescence.
>>
>>                                     Guy
>>
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>     http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
>> Sent: Monday, 31 October 2011 11:08 PM
>> To: [hidden email]
>> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>>
>>
>> Quoting Guy Cox <[hidden email]>:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Daniel White wrote:
>>>
>>> " in a confocal you throw away most of the signal, as its out of focus.
>>> So as a result the images are often very noisy.  "
>>>
>>> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>>> noise, not signal.  If you have no SA (and, honestly, if you are
>>> seriously interested in high-resolution imaging that should be a given)
>>> then a confocal microscope with the pinhole set at 1 Airy diameter
>>> throws away no signal at all.  So why are confocal images often noisy?
>>> Well, it's just statistics.  If you take a wide-field image with a 1
>>> second exposure each point is exposed for one second.  If you take a
>>> confocal image at 512 x 512 for 1 second then each point is exposed for
>>> ~4 microseconds.  The difference is rather substantial ...
>>>
>>>                                           Guy
>>>
>>> Optical Imaging Techniques in Cell Biology
>>> by Guy Cox    CRC Press / Taylor & Francis
>>>     http://www.guycox.com/optical.htm
>>> ______________________________________________
>>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>>> Australian Centre for Microscopy & Microanalysis,
>>> Madsen Building F09, University of Sydney, NSW 2006
>>>
>>> Phone [phone]     Fax [phone]
>>>             Mobile [phone]
>>> ______________________________________________
>>>      http://www.guycox.net
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>> On Behalf Of daniel white
>>> Sent: Monday, 31 October 2011 8:30 PM
>>> To: [hidden email]
>>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Peter,
>>>
>>> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>>> wrote:
>>>
>>>>
>>>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>>> From:    Peter Werner <[hidden email]>
>>>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>>
>>>> An interesting point was made here by Jim Pawley:
>>>>
>>>>> I agree that sampling a bit higher than Nyquist never hurts,
>>>>> especially if you deconvolve (as you always should), but I think
>>>>> that it is a mistake to think that one can "separate" out the noise
>>>>> by decon. I think that noise is pretty fundamental.
>>>>
>>>> I had always heard that if you're doing confocal microscopy, at least
>>>
>>>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>>>> deconvolution was superfluous, because you shouldn't be getting out of
>>>
>>>> focus light. So what is gained by deconvolution when one is sampling
>>>> voxel by voxel?
>>>
>>> in a confocal you throw away most of the signal, as its out of focus.
>>> So as a result the images are often very noisy.
>>> Good contrast.... but high Poisson distributed photon shot noise
>>> from only measuring a handful of photons.
>>>
>>> So usually one needs to do something about that noise...
>>> we want to separate the real signal from the noise.
>>>
>>> Often a Gaussian or mean filter is applied... which suppresses the noise
>>>
>>> by smoothing it out... but it also smooths the real signal, so
>>> effectively you lose
>>> the contrast and resolution that was the whole point of doing confocal.
>>>
>>> The smart way to suppress the noise, but keep the contrast and
>>> resolution
>>> is to do deconvolution.
>>> Deconvolution using a max likelyhood method uses the known shape of the
>>> PSF
>>> to make a best guess model of the real fluorophore distribution in the
>>> sample.
>>> You tell the deconvolution algorithm how noisy the image is (you have to
>>> guess
>>> unless you take 2 images and measure it)
>>> then it attempts to throw out the noise and keep the real signal,
>>> resolution and contrast intact.
>>>
>>> D
>>>
>>>>
>>>> Peter G. Werner
>>>> Merritt College Microscopy Program
>>>
>>> Dr. Daniel James White BSc. (Hons.) PhD
>>>
>>> Leader - Image Processing Facility,
>>> Senior Microscopist,
>>> Light Microscopy Facility.
>>>
>>> Max Planck Institute of Molecular Cell Biology and Genetics
>>> Pfotenhauerstrasse 108
>>> 01307 DRESDEN
>>> Germany
>>>
>>> [phone] (German Mobile)
>>> [phone] (Work phone at MPI-CBG)
>>> [phone] (Fax MPI-CBG LMF)
>>> chalkie666                                   Skype
>>> http://www.bioimagexd.net    BioImageXD
>>> http://fiji.sc                                       Fiji -  is just ImageJ
>>> (Batteries Included)
>>> http://www.chalkie.org.uk            Dan's Homepages
>>> https://ifn.mpi-cbg.de                       Biopolis Dresden Imaging
>>> Platform (BioDIP)
>>> dan (at) chalkie.org.uk
>>> ( white (at) mpi-cbg.de )
>>>
>>> -----
>>> No virus found in this message.
>>> Checked by AVG - www.avg.com
>>> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>>
>>
>>
>>
>> Jeremy Adler
>> IGP
>> Rudbeckslaboratoriet
>> Daghammersköljdsväg 20
>> 751 85 Uppsala
>> Sweden
>>
>> 0046 (0)18 471 4607
>>
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
>> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
>> Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11
>

[Jeremy Adler-4]

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

Sorry Guy to reuse your unnecessarily terse comment, it is you who are wrong.

Multiphoton imaging clearly does limit the origins of any fluorescence  
but does not address the original problem, Poisson noise, about why  
confocal images appear noisy - lack of in focus photons, which you  
correctly pointed out.
Deconvolution of widefield images does address Poisson noise since it  
efficiently uses all detectable photons and therefore is worthwhile. A  
deconvolved widefield image will often be preferable to a confocal or  
multiphoton image.
There is also a semantic question about what constitutes noise and if  
unwanted out of focus photons can be returned to their origins then  
they cease to be noise and become signal.


Quoting Guy Cox <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> " The photons that are rejected by a pinhole usually come from
> fluorophores within the specimen - deconvolution can be viewed as an
> attempt to improve images by putting photons back to where they
> probably originated, rather than to just reject them. Deconvolution
> makes a more efficient use of the emitted photons.
>
> It is therefore possible to obtain an image by deconvolving a
> widefield z series that, because of photobleaching and rejection of
> photons by a pinhole, cannot be obtained from a confocal, even if
> acquisition time was not the limiting constraint.
>
> Only in the narrowest sense, when only a single optical section is
> required, is Guy correct in regarding out of focus fluorescence as
> noise - perhaps signal of unwanted origin."
>
> WRONG!!  Yes, of course the photons come from fluorophores within  
> the specimen.  Where else could they come from?  But they don't come  
> from where we are looking at and so they cannot be assigned to the  
> plane we are imaging.  They belong in a different plane and should  
> be assigned there.  And in a confocal stack that is exactly what  
> will happen.  So of course there is wasted fluorescence - and if we  
> want to avoid this the answer is rather simple - use 2-photon. Then  
> there is NO out of plane fluorescence.
>
>                                      Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List  
> [mailto:[hidden email]] On Behalf Of Jeremy Adler
> Sent: Monday, 31 October 2011 11:08 PM
> To: [hidden email]
> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
>
>
> Quoting Guy Cox <[hidden email]>:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Daniel White wrote:
>>
>> " in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.  "
>>
>> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>> noise, not signal.  If you have no SA (and, honestly, if you are
>> seriously interested in high-resolution imaging that should be a given)
>> then a confocal microscope with the pinhole set at 1 Airy diameter
>> throws away no signal at all.  So why are confocal images often noisy?
>> Well, it's just statistics.  If you take a wide-field image with a 1
>> second exposure each point is exposed for one second.  If you take a
>> confocal image at 512 x 512 for 1 second then each point is exposed for
>> ~4 microseconds.  The difference is rather substantial ...
>>
>>                                            Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>              Mobile [phone]
>> ______________________________________________
>>       http://www.guycox.net
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of daniel white
>> Sent: Monday, 31 October 2011 8:30 PM
>> To: [hidden email]
>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Peter,
>>
>> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>> wrote:
>>
>>>
>>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>> From:    Peter Werner <[hidden email]>
>>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>> An interesting point was made here by Jim Pawley:
>>>
>>>> I agree that sampling a bit higher than Nyquist never hurts,
>>>> especially if you deconvolve (as you always should), but I think
>>>> that it is a mistake to think that one can "separate" out the noise
>>>> by decon. I think that noise is pretty fundamental.
>>>
>>> I had always heard that if you're doing confocal microscopy, at least
>>
>>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>>> deconvolution was superfluous, because you shouldn't be getting out of
>>
>>> focus light. So what is gained by deconvolution when one is sampling
>>> voxel by voxel?
>>
>> in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.
>> Good contrast.... but high Poisson distributed photon shot noise
>> from only measuring a handful of photons.
>>
>> So usually one needs to do something about that noise...
>> we want to separate the real signal from the noise.
>>
>> Often a Gaussian or mean filter is applied... which suppresses the noise
>>
>> by smoothing it out... but it also smooths the real signal, so
>> effectively you lose
>> the contrast and resolution that was the whole point of doing confocal.
>>
>> The smart way to suppress the noise, but keep the contrast and
>> resolution
>> is to do deconvolution.
>> Deconvolution using a max likelyhood method uses the known shape of the
>> PSF
>> to make a best guess model of the real fluorophore distribution in the
>> sample.
>> You tell the deconvolution algorithm how noisy the image is (you have to
>> guess
>> unless you take 2 images and measure it)
>> then it attempts to throw out the noise and keep the real signal,
>> resolution and contrast intact.
>>
>> D
>>
>>>
>>> Peter G. Werner
>>> Merritt College Microscopy Program
>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>>
>> Leader - Image Processing Facility,
>> Senior Microscopist,
>> Light Microscopy Facility.
>>
>> Max Planck Institute of Molecular Cell Biology and Genetics
>> Pfotenhauerstrasse 108
>> 01307 DRESDEN
>> Germany
>>
>> [phone] (German Mobile)
>> [phone] (Work phone at MPI-CBG)
>> [phone] (Fax MPI-CBG LMF)
>> chalkie666 Skype
>> http://www.bioimagexd.net  BioImageXD
>> http://fiji.sc                                        Fiji -  is just ImageJ
>> (Batteries Included)
>> http://www.chalkie.org.uk                Dan's Homepages
>> https://ifn.mpi-cbg.de  Biopolis Dresden Imaging
>> Platform (BioDIP)
>> dan (at) chalkie.org.uk
>> ( white (at) mpi-cbg.de )
>>
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
>> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>
>
>
>
> Jeremy Adler
> IGP
> Rudbeckslaboratoriet
> Daghammersköljdsväg 20
> 751 85 Uppsala
> Sweden
>
> 0046 (0)18 471 4607
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[mmodel]

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

In deciding which image is "better", we can't go just by their visual attractiveness, we should also consider whether the image correctly represents the object. We have done such a study a couple years ago, and concluded that when spherical aberration is present, deconvolution improves the image, but there is still a lot of residual axial elongation. There we stopped, and I don't know if the resulting distortion is better or worse than confocal restoration or deconvolved confocal images, perhaps someone will find it out one day.

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
Sent: Monday, October 31, 2011 9:53 AM
To: [hidden email]
Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)

*****
To join, leave or search the confocal microscopy listserv, go to:
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Sorry Guy to reuse your unnecessarily terse comment, it is you who are wrong.

Multiphoton imaging clearly does limit the origins of any fluorescence  
but does not address the original problem, Poisson noise, about why  
confocal images appear noisy - lack of in focus photons, which you  
correctly pointed out.
Deconvolution of widefield images does address Poisson noise since it  
efficiently uses all detectable photons and therefore is worthwhile. A  
deconvolved widefield image will often be preferable to a confocal or  
multiphoton image.
There is also a semantic question about what constitutes noise and if  
unwanted out of focus photons can be returned to their origins then  
they cease to be noise and become signal.


Quoting Guy Cox <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> " The photons that are rejected by a pinhole usually come from
> fluorophores within the specimen - deconvolution can be viewed as an
> attempt to improve images by putting photons back to where they
> probably originated, rather than to just reject them. Deconvolution
> makes a more efficient use of the emitted photons.
>
> It is therefore possible to obtain an image by deconvolving a
> widefield z series that, because of photobleaching and rejection of
> photons by a pinhole, cannot be obtained from a confocal, even if
> acquisition time was not the limiting constraint.
>
> Only in the narrowest sense, when only a single optical section is
> required, is Guy correct in regarding out of focus fluorescence as
> noise - perhaps signal of unwanted origin."
>
> WRONG!!  Yes, of course the photons come from fluorophores within  
> the specimen.  Where else could they come from?  But they don't come  
> from where we are looking at and so they cannot be assigned to the  
> plane we are imaging.  They belong in a different plane and should  
> be assigned there.  And in a confocal stack that is exactly what  
> will happen.  So of course there is wasted fluorescence - and if we  
> want to avoid this the answer is rather simple - use 2-photon. Then  
> there is NO out of plane fluorescence.
>
>                                      Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List  
> [mailto:[hidden email]] On Behalf Of Jeremy Adler
> Sent: Monday, 31 October 2011 11:08 PM
> To: [hidden email]
> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
>
>
> Quoting Guy Cox <[hidden email]>:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Daniel White wrote:
>>
>> " in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.  "
>>
>> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>> noise, not signal.  If you have no SA (and, honestly, if you are
>> seriously interested in high-resolution imaging that should be a given)
>> then a confocal microscope with the pinhole set at 1 Airy diameter
>> throws away no signal at all.  So why are confocal images often noisy?
>> Well, it's just statistics.  If you take a wide-field image with a 1
>> second exposure each point is exposed for one second.  If you take a
>> confocal image at 512 x 512 for 1 second then each point is exposed for
>> ~4 microseconds.  The difference is rather substantial ...
>>
>>                                            Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>              Mobile [phone]
>> ______________________________________________
>>       http://www.guycox.net
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of daniel white
>> Sent: Monday, 31 October 2011 8:30 PM
>> To: [hidden email]
>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Peter,
>>
>> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>> wrote:
>>
>>>
>>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>> From:    Peter Werner <[hidden email]>
>>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>> An interesting point was made here by Jim Pawley:
>>>
>>>> I agree that sampling a bit higher than Nyquist never hurts,
>>>> especially if you deconvolve (as you always should), but I think
>>>> that it is a mistake to think that one can "separate" out the noise
>>>> by decon. I think that noise is pretty fundamental.
>>>
>>> I had always heard that if you're doing confocal microscopy, at least
>>
>>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>>> deconvolution was superfluous, because you shouldn't be getting out of
>>
>>> focus light. So what is gained by deconvolution when one is sampling
>>> voxel by voxel?
>>
>> in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.
>> Good contrast.... but high Poisson distributed photon shot noise
>> from only measuring a handful of photons.
>>
>> So usually one needs to do something about that noise...
>> we want to separate the real signal from the noise.
>>
>> Often a Gaussian or mean filter is applied... which suppresses the noise
>>
>> by smoothing it out... but it also smooths the real signal, so
>> effectively you lose
>> the contrast and resolution that was the whole point of doing confocal.
>>
>> The smart way to suppress the noise, but keep the contrast and
>> resolution
>> is to do deconvolution.
>> Deconvolution using a max likelyhood method uses the known shape of the
>> PSF
>> to make a best guess model of the real fluorophore distribution in the
>> sample.
>> You tell the deconvolution algorithm how noisy the image is (you have to
>> guess
>> unless you take 2 images and measure it)
>> then it attempts to throw out the noise and keep the real signal,
>> resolution and contrast intact.
>>
>> D
>>
>>>
>>> Peter G. Werner
>>> Merritt College Microscopy Program
>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>>
>> Leader - Image Processing Facility,
>> Senior Microscopist,
>> Light Microscopy Facility.
>>
>> Max Planck Institute of Molecular Cell Biology and Genetics
>> Pfotenhauerstrasse 108
>> 01307 DRESDEN
>> Germany
>>
>> [phone] (German Mobile)
>> [phone] (Work phone at MPI-CBG)
>> [phone] (Fax MPI-CBG LMF)
>> chalkie666 Skype
>> http://www.bioimagexd.net  BioImageXD
>> http://fiji.sc                                        Fiji -  is just ImageJ
>> (Batteries Included)
>> http://www.chalkie.org.uk                Dan's Homepages
>> https://ifn.mpi-cbg.de  Biopolis Dresden Imaging
>> Platform (BioDIP)
>> dan (at) chalkie.org.uk
>> ( white (at) mpi-cbg.de )
>>
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
>> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>
>
>
>
> Jeremy Adler
> IGP
> Rudbeckslaboratoriet
> Daghammersköljdsväg 20
> 751 85 Uppsala
> Sweden
>
> 0046 (0)18 471 4607
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[Lutz Schaefer]

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

Surprisingly, as this discussion repeats itself on the server several times
over and over, there are still misunderstandings. First of all,
deconvolution is nothing more than an attempt to invert a model of the
forward imaging problem. This model almost never includes that out-of-focus
light is considered noise in a statistical sense. Instead it is considered
the result of a convolution operation with the PSF, where its inverse
reassigns this light back to the focal plane, contrary to Guy's statement.
Noise, on the other hand is often additionally included in the forward model
for both Gauss and Poisson distributions - resulting in an inverse solution
(estimate) known as maximum likelihood. Advanced deconvolution systems can
optimize for both at the same time as the Gaussian camera read-out noise
will always be present, perhaps except for rare cases were the photon noise
dominates by orders of magnitude.

Best 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: [phone]
Email:     [hidden email]
___________________________________

--------------------------------------------------
From: "Guy Cox" <[hidden email]>
Sent: Monday, October 31, 2011 08:47
To: <[hidden email]>
Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> " The photons that are rejected by a pinhole usually come from
> fluorophores within the specimen - deconvolution can be viewed as an
> attempt to improve images by putting photons back to where they
> probably originated, rather than to just reject them. Deconvolution
> makes a more efficient use of the emitted photons.
>
> It is therefore possible to obtain an image by deconvolving a
> widefield z series that, because of photobleaching and rejection of
> photons by a pinhole, cannot be obtained from a confocal, even if
> acquisition time was not the limiting constraint.
>
> Only in the narrowest sense, when only a single optical section is
> required, is Guy correct in regarding out of focus fluorescence as
> noise - perhaps signal of unwanted origin."
>
> WRONG!!  Yes, of course the photons come from fluorophores within the
> specimen.  Where else could they come from?  But they don't come from
> where we are looking at and so they cannot be assigned to the plane we are
> imaging.  They belong in a different plane and should be assigned there.
> And in a confocal stack that is exactly what will happen.  So of course
> there is wasted fluorescence - and if we want to avoid this the answer is
> rather simple - use 2-photon. Then there is NO out of plane fluorescence.
>
>                                     Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Jeremy Adler
> Sent: Monday, 31 October 2011 11:08 PM
> To: [hidden email]
> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
>
>
> Quoting Guy Cox <[hidden email]>:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Daniel White wrote:
>>
>> " in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.  "
>>
>> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>> noise, not signal.  If you have no SA (and, honestly, if you are
>> seriously interested in high-resolution imaging that should be a given)
>> then a confocal microscope with the pinhole set at 1 Airy diameter
>> throws away no signal at all.  So why are confocal images often noisy?
>> Well, it's just statistics.  If you take a wide-field image with a 1
>> second exposure each point is exposed for one second.  If you take a
>> confocal image at 512 x 512 for 1 second then each point is exposed for
>> ~4 microseconds.  The difference is rather substantial ...
>>
>>                                            Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>              Mobile [phone]
>> ______________________________________________
>>       http://www.guycox.net
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of daniel white
>> Sent: Monday, 31 October 2011 8:30 PM
>> To: [hidden email]
>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Peter,
>>
>> On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>> wrote:
>>
>>>
>>> Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>> From:    Peter Werner <[hidden email]>
>>> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>> An interesting point was made here by Jim Pawley:
>>>
>>>> I agree that sampling a bit higher than Nyquist never hurts,
>>>> especially if you deconvolve (as you always should), but I think
>>>> that it is a mistake to think that one can "separate" out the noise
>>>> by decon. I think that noise is pretty fundamental.
>>>
>>> I had always heard that if you're doing confocal microscopy, at least
>>
>>> point-scanning confocal with a pinhole size of 1AU or smaller, that
>>> deconvolution was superfluous, because you shouldn't be getting out of
>>
>>> focus light. So what is gained by deconvolution when one is sampling
>>> voxel by voxel?
>>
>> in a confocal you throw away most of the signal, as its out of focus.
>> So as a result the images are often very noisy.
>> Good contrast.... but high Poisson distributed photon shot noise
>> from only measuring a handful of photons.
>>
>> So usually one needs to do something about that noise...
>> we want to separate the real signal from the noise.
>>
>> Often a Gaussian or mean filter is applied... which suppresses the noise
>>
>> by smoothing it out... but it also smooths the real signal, so
>> effectively you lose
>> the contrast and resolution that was the whole point of doing confocal.
>>
>> The smart way to suppress the noise, but keep the contrast and
>> resolution
>> is to do deconvolution.
>> Deconvolution using a max likelyhood method uses the known shape of the
>> PSF
>> to make a best guess model of the real fluorophore distribution in the
>> sample.
>> You tell the deconvolution algorithm how noisy the image is (you have to
>> guess
>> unless you take 2 images and measure it)
>> then it attempts to throw out the noise and keep the real signal,
>> resolution and contrast intact.
>>
>> D
>>
>>>
>>> Peter G. Werner
>>> Merritt College Microscopy Program
>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>>
>> Leader - Image Processing Facility,
>> Senior Microscopist,
>> Light Microscopy Facility.
>>
>> Max Planck Institute of Molecular Cell Biology and Genetics
>> Pfotenhauerstrasse 108
>> 01307 DRESDEN
>> Germany
>>
>> [phone] (German Mobile)
>> [phone] (Work phone at MPI-CBG)
>> [phone] (Fax MPI-CBG LMF)
>> chalkie666 Skype
>> http://www.bioimagexd.net BioImageXD
>> http://fiji.sc Fiji -  is just ImageJ
>> (Batteries Included)
>> http://www.chalkie.org.uk Dan's Homepages
>> https://ifn.mpi-cbg.de Biopolis Dresden Imaging
>> Platform (BioDIP)
>> dan (at) chalkie.org.uk
>> ( white (at) mpi-cbg.de )
>>
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
>> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>
>
>
>
> Jeremy Adler
> IGP
> Rudbeckslaboratoriet
> Daghammersköljdsväg 20
> 751 85 Uppsala
> Sweden
>
> 0046 (0)18 471 4607
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11

[James Mansfield]

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

Hi,

I'm not sure I like calling out-of-focal-plane photons "noise". They are not noise, they are just signals from places where you don't happen to want them at the moment.

To take a set of terms from another field, the military hyperspectral imaging crowd (who are generally trying to isolate the signal from a tank or other military object from the background of normal non-military objects) have come up with:

Noise: random or instrument signals that are an artifact of the way you collected the data
Signal: the signal you actually care about
Clutter: other (real) signals that interfering with your ability to see the signal

Depending on the sample, either "noise" or "clutter" are the limiting factors. For many samples looked at in confocal, noise is the limiting factor. For many highly autofluorescent tissue samples, clutter is the limiting factor and needs to be dealt with differently than random noise.

Jim



James R. Mansfield
Director, Tissue Analysis Applications
Caliper Life Sciences, Inc.
68 Elm Street, Hopkinton, MA 01748
Office: [phone]
Cell: [phone]
Email: [hidden email]
www.caliperls.com
www.cri-inc.com

Need to send me large files? Use my YouSendIt DropBox:
https://dropbox.yousendit.com/Mansfield-Dropbox


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Monday, October 31, 2011 7:21 AM
To: [hidden email]
Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)

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

Daniel White wrote:

" in a confocal you throw away most of the signal, as its out of focus.
So as a result the images are often very noisy.  "

This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
noise, not signal.  If you have no SA (and, honestly, if you are
seriously interested in high-resolution imaging that should be a given)
then a confocal microscope with the pinhole set at 1 Airy diameter
throws away no signal at all.  So why are confocal images often noisy?
Well, it's just statistics.  If you take a wide-field image with a 1
second exposure each point is exposed for one second.  If you take a
confocal image at 512 x 512 for 1 second then each point is exposed for
~4 microseconds.  The difference is rather substantial ...

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of daniel white
Sent: Monday, 31 October 2011 8:30 PM
To: [hidden email]
Subject: Deconvolution of Confocal Images? (was: Airy Units)

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

Hi Peter,

On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
wrote:

>
> Date:    Sun, 30 Oct 2011 13:09:10 -0700
> From:    Peter Werner <[hidden email]>
> Subject: Deconvolution of Confocal Images? (was: Airy Units)
>
> An interesting point was made here by Jim Pawley:
>
>> I agree that sampling a bit higher than Nyquist never hurts,
>> especially if you deconvolve (as you always should), but I think
>> that it is a mistake to think that one can "separate" out the noise
>> by decon. I think that noise is pretty fundamental.
>
> I had always heard that if you're doing confocal microscopy, at least

> point-scanning confocal with a pinhole size of 1AU or smaller, that
> deconvolution was superfluous, because you shouldn't be getting out of

> focus light. So what is gained by deconvolution when one is sampling
> voxel by voxel?

in a confocal you throw away most of the signal, as its out of focus.
So as a result the images are often very noisy.
Good contrast.... but high Poisson distributed photon shot noise
from only measuring a handful of photons.

So usually one needs to do something about that noise...
we want to separate the real signal from the noise.

Often a Gaussian or mean filter is applied... which suppresses the noise

by smoothing it out... but it also smooths the real signal, so
effectively you lose
the contrast and resolution that was the whole point of doing confocal.

The smart way to suppress the noise, but keep the contrast and
resolution
is to do deconvolution.
Deconvolution using a max likelyhood method uses the known shape of the
PSF
to make a best guess model of the real fluorophore distribution in the
sample.
You tell the deconvolution algorithm how noisy the image is (you have to
guess
unless you take 2 images and measure it)
then it attempts to throw out the noise and keep the real signal,
resolution and contrast intact.

D

>
> Peter G. Werner
> Merritt College Microscopy Program

Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666                                      Skype
http://www.bioimagexd.net       BioImageXD
http://fiji.sc                                  Fiji -  is just ImageJ
(Batteries Included)
http://www.chalkie.org.uk               Dan's Homepages
https://ifn.mpi-cbg.de                  Biopolis Dresden Imaging
Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

-----
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Checked by AVG - www.avg.com
Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11

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[James Pawley]

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>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Sorry Guy to reuse your unnecessarily terse comment, it is you who are wrong.
>
>Multiphoton imaging clearly does limit the
>origins of any fluorescence but does not address
>the original problem, Poisson noise, about why
>confocal images appear noisy - lack of in focus
>photons, which you correctly pointed out.
>Deconvolution of widefield images does address
>Poisson noise since it efficiently uses all
>detectable photons and therefore is worthwhile.
>A deconvolved widefield image will often be
>preferable to a confocal or multiphoton image.
>There is also a semantic question about what
>constitutes noise and if unwanted out of focus
>photons can be returned to their origins then
>they cease to be noise and become signal.
>

Hi again,

Let's recognize that decon is at its base a
mathematical process relating to continuous,
mathematical functions. Here is works perfectly!
Therefore, that although the idea that "inversion
yields microscope truth" has appeal, it has
little to do with Fluorescence Microscopy,
especially that of living cells where the data is
discontinuous (pixellated) and where Poisson
noise is extremely important.

Maybe we need some numbers here. If we assume a
CCD read noise of +/- 5e/pixel, it will be
overshadowed by Poisson noise for any signal
level above 5x5= 25e/pixel. I submit that few
pixels in a widefield CCD data set record a
signal representing less than 25e (not
necessarily 25 counts in the memory but that
number multiplied by the gain-factor of the
camera to represent e/pixel). So Poisson Noise is
always dominant.

On the other hand, Gaussian noise is a lot easier
to model and, if there is a lot of out-of-focus
light (so that the in-plane contrast is say only
1% or even 10% of the total, then most pixels
have the almost the same number of electrons and
the square-root of these various numbers will
vary even less. This is the rationale for
assuming that Poisson Noise to be approximated by
a Gaussian without too much problem in widefield
decon.

As to "which is best":

I start by assuming that all 3D fluorescence
microscope data should be deconvolved.
Therefore, the question comes down to which is
the best method of data collection. Widefield
records some near-focus data that might be useful
and that confocal (may) exclude (depending on the
pinhole size). However, if there are bright
features farther from the focus plane, then they
will produce recorded photons that are probably
less useful. In the end it is all a matter of S/N
and this means statistics.

As a result. the decision depends on the geometry
of the specimen stain distribution and in
particular, the extent to which the greater QE of
the CCD (used in widefield. Greater QE reduces
Poisson Noise on the same light signal.),
compared to that of the PMT (used in confocal.
Depending on wavelength, the PMT is about 10x
worse than the CCD but getting better with the
new photon-counting, GaAsP detectors.), is offset
by the Poisson Noise introduced into the
widefield image by bright, out-of-focus features.
i.e., WF/Decon that does very well on
tissue-culture slides (at the UBC Course, for
instance) may have more problems, with embryos if
(and only if) they contain many stained features
in depth.)

Assuming that the acceptance angle of the
objective is 60 deg, the signal recorded in a
pixel of the size needed to Nyquist sample an
in-focus point object, will be about 80x lower
when the point object is only 1µm out of focus
than when it was in focus. This seems to indicate
that it will be hard to record any useful
(in-focus?) signal from features that are more
than 2 or 3 µm away from the focus plane.

However,  in WF, large, bright features much
farther from the focus plane will still produced
significant detectable signal (and its associated
Poisson Noise) and it is hard to see how this can
ever be thought useful.

Confocal and multiphoton will not record any signal from such features.

Personally, I feel that the "put the photons back
where they came from" wording is extremely
misleading.

Yes, we think that the processed 3D image
resembles the original object more closely than
does the raw data, but we seldom have any way to
check this. Essentially, the decon process is no
more immune to error than any other
extrapolation. As the data going into it noisy,
the output also has an error rate and the mere
fact that it looks convincing should not blind us
to the need to remember that its validity rests
on  assumptions that may not be valid.

For instance, decon assumes that the PSF is the
same over the whole field of view and at every
successive plane recorded. Neither of these
assumptions are "true" in any general sense (see
Handbook Chapters 11 and 20). An extreme example
is shown in Scanning, 24: 241-246 and also
Chapter 35. Here, the presence of the nucleus in
a living cheek cell not only greatly distorts the
PSF when recording features on its far side, it
also can displace them up to 6 micrometers. I
submit that there is no practical and general
method for "deconvolving" out this sort of
distortion.

In addition, 3D widefield data will include only
part of the light from emitted by features near
the edges (or even just outside) of the data
stack and hence this edge volume cannot be
deconvolved. The larger the PSF used, the larger
this volume of incomplete data will be.

And when time or photosensitivity constraints
limit one to collecting only 2D vs time data, the
confocal/multiphoton approach has much to offer,

So, can I suggest that we be content to be glad
that we have all these nice microscopes without
feeling that we have to win some sort of horse
race?

Also we need to we keep in mind that, as optimal
imaging conditions seldom overlap with optimal
cell viability, we need to remain cautious when
drawing conclusions from the high-resolution
aspects of any of the data that these techniques
produce.

Ciao,

JP

***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953              
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 10-22, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/        Applications accepted after 11/15/12
               "If it ain't diffraction, it must be statistics." Anon.

>Quoting Guy Cox <[hidden email]>:
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>" The photons that are rejected by a pinhole usually come from
>>fluorophores within the specimen - deconvolution can be viewed as an
>>attempt to improve images by putting photons back to where they
>>probably originated, rather than to just reject them. Deconvolution
>>makes a more efficient use of the emitted photons.
>>
>>It is therefore possible to obtain an image by deconvolving a
>>widefield z series that, because of photobleaching and rejection of
>>photons by a pinhole, cannot be obtained from a confocal, even if
>>acquisition time was not the limiting constraint.
>>
>>Only in the narrowest sense, when only a single optical section is
>>required, is Guy correct in regarding out of focus fluorescence as
>>noise - perhaps signal of unwanted origin."
>>
>>WRONG!!  Yes, of course the photons come from
>>fluorophores within the specimen.  Where else
>>could they come from?  But they don't come from
>>where we are looking at and so they cannot be
>>assigned to the plane we are imaging.  They
>>belong in a different plane and should be
>>assigned there.  And in a confocal stack that
>>is exactly what will happen.  So of course
>>there is wasted fluorescence - and if we want
>>to avoid this the answer is rather simple - use
>>2-photon. Then there is NO out of plane
>>fluorescence.
>>
>>                                      Guy
>>
>>
>>Optical Imaging Techniques in Cell Biology
>>by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>>______________________________________________
>>Associate Professor Guy Cox, MA, DPhil(Oxon)
>>Australian Centre for Microscopy & Microanalysis,
>>Madsen Building F09, University of Sydney, NSW 2006
>>
>>Phone [phone]     Fax [phone]
>>              Mobile [phone]
>>______________________________________________
>>       http://www.guycox.net
>>
>>
>>
>>-----Original Message-----
>>From: Confocal Microscopy List
>>[mailto:[hidden email]] On
>>Behalf Of Jeremy Adler
>>Sent: Monday, 31 October 2011 11:08 PM
>>To: [hidden email]
>>Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>
>>
>>
>>Quoting Guy Cox <[hidden email]>:
>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>Daniel White wrote:
>>>
>>>" in a confocal you throw away most of the signal, as its out of focus.
>>>So as a result the images are often very noisy.  "
>>>
>>>This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>>>noise, not signal.  If you have no SA (and, honestly, if you are
>>>seriously interested in high-resolution imaging that should be a given)
>>>then a confocal microscope with the pinhole set at 1 Airy diameter
>>>throws away no signal at all.  So why are confocal images often noisy?
>>>Well, it's just statistics.  If you take a wide-field image with a 1
>>>second exposure each point is exposed for one second.  If you take a
>>>confocal image at 512 x 512 for 1 second then each point is exposed for
>>>~4 microseconds.  The difference is rather substantial ...
>>>
>>>                                            Guy
>>>
>>>Optical Imaging Techniques in Cell Biology
>>>by Guy Cox    CRC Press / Taylor & Francis
>>>      http://www.guycox.com/optical.htm
>>>______________________________________________
>>>Associate Professor Guy Cox, MA, DPhil(Oxon)
>>>Australian Centre for Microscopy & Microanalysis,
>>>Madsen Building F09, University of Sydney, NSW 2006
>>>
>>>Phone [phone]     Fax [phone]
>>>              Mobile [phone]
>>>______________________________________________
>>>       http://www.guycox.net
>>>
>>>
>>>-----Original Message-----
>>>From: Confocal Microscopy List [mailto:[hidden email]]
>>>On Behalf Of daniel white
>>>Sent: Monday, 31 October 2011 8:30 PM
>>>To: [hidden email]
>>>Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>Hi Peter,
>>>
>>>On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>>>wrote:
>>>
>>>>
>>>>Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>>>From:    Peter Werner <[hidden email]>
>>>>Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>>
>>>>An interesting point was made here by Jim Pawley:
>>>>
>>>>>I agree that sampling a bit higher than Nyquist never hurts,
>>>>>especially if you deconvolve (as you always should), but I think
>>>>>that it is a mistake to think that one can "separate" out the noise
>>>>>by decon. I think that noise is pretty fundamental.
>>>>
>>>>I had always heard that if you're doing confocal microscopy, at least
>>>
>>>>point-scanning confocal with a pinhole size of 1AU or smaller, that
>>>>deconvolution was superfluous, because you shouldn't be getting out of
>>>
>>>>focus light. So what is gained by deconvolution when one is sampling
>>>>voxel by voxel?
>>>
>>>in a confocal you throw away most of the signal, as its out of focus.
>>>So as a result the images are often very noisy.
>>>Good contrast.... but high Poisson distributed photon shot noise
>>>from only measuring a handful of photons.
>>>
>>>So usually one needs to do something about that noise...
>>>we want to separate the real signal from the noise.
>>>
>>>Often a Gaussian or mean filter is applied... which suppresses the noise
>>>
>>>by smoothing it out... but it also smooths the real signal, so
>>>effectively you lose
>>>the contrast and resolution that was the whole point of doing confocal.
>>>
>>>The smart way to suppress the noise, but keep the contrast and
>>>resolution
>>>is to do deconvolution.
>>>Deconvolution using a max likelyhood method uses the known shape of the
>>>PSF
>>>to make a best guess model of the real fluorophore distribution in the
>>>sample.
>>>You tell the deconvolution algorithm how noisy the image is (you have to
>>>guess
>>>unless you take 2 images and measure it)
>>>then it attempts to throw out the noise and keep the real signal,
>>>resolution and contrast intact.
>>>
>>>D
>>>
>>>>
>>>>Peter G. Werner
>>>>Merritt College Microscopy Program
>>>
>>>Dr. Daniel James White BSc. (Hons.) PhD
>>>
>>>Leader - Image Processing Facility,
>>>Senior Microscopist,
>>>Light Microscopy Facility.
>>>
>>>Max Planck Institute of Molecular Cell Biology and Genetics
>>>Pfotenhauerstrasse 108
>>>01307 DRESDEN
>>>Germany
>>>
>>>[phone] (German Mobile)
>>>[phone] (Work phone at MPI-CBG)
>>>[phone] (Fax MPI-CBG LMF)
>>>chalkie666 Skype
>>>http://www.bioimagexd.net        BioImageXD
>>>http://fiji.sc                                        Fiji -  is just ImageJ
>>>(Batteries Included)
>>>http://www.chalkie.org.uk                Dan's Homepages
>>>https://ifn.mpi-cbg.de                        Biopolis Dresden Imaging
>>>Platform (BioDIP)
>>>dan (at) chalkie.org.uk
>>>( white (at) mpi-cbg.de )
>>>
>>>-----
>>>No virus found in this message.
>>>Checked by AVG - www.avg.com
>>>Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>>
>>
>>
>>
>>Jeremy Adler
>>IGP
>>Rudbeckslaboratoriet
>>Daghammersköljdsväg 20
>>751 85 Uppsala
>>Sweden
>>
>>0046 (0)18 471 4607
>>
>>-----
>>No virus found in this message.
>>Checked by AVG - www.avg.com
>>Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>-----
>>No virus found in this message.
>>Checked by AVG - www.avg.com
>>Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11
>>
>
>
>
>Jeremy Adler
>IGP
>Rudbeckslaboratoriet
>Daghammersköljdsväg 20
>751 85 Uppsala
>Sweden
>
>0046 (0)18 471 4607

--
***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953              
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 10-22, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/        Applications accepted after 11/15/12
               "If it ain't diffraction, it must be statistics." Anon.

[Guy Cox-2]

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

Jim,

        My comment was unnecessarily terse, and I apologise for that.  But it does get me cranky when people claim there's some value in the out of focus light.  You yourself pointed out that this is crazy, some time ago.  Just one micrometre from the focal plane the cone of light from a high NA lens is 6µm in diameter.  Go much further and it's everwhere!  (This is just repeating what you have said).   We can of course work out the intensity distribution expected 1µm from a bright point, and subtract it from that plane (that's deconvolution, or part of it).  But unless our sample is trivially sparse we can't predict the bright spot from its ghost at any substantial defocus.  (And if our sample is so sparse conventional resolution doesn't apply - that's STORM).  So we are not using all detectable photons in any meaningful sense.  What is more important, we are not using all excited fluorescence, since each fluorochrome molecule has a finite life.  

        Confocal images will always be noisier than WF unless we scan for a very long time - we all seem to be agreed on that.  But both are exciting fluorescence outside the focal plane.  Multiphoton does not - we actually CAN make use of every detected photon.  We will need patience, granted, but if people are prepared to take hours acquiring a STORM or PALM dataset maybe they should think about taking several minutes to acquire a multiphoton one?  (And then, maybe, deconvolving it).  

        (I fear that my µ symbols may not make it through the list server, so where you see  µm it means micrometres).

                                      Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Tuesday, 1 November 2011 7:17 AM
To: [hidden email]
Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)

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

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Sorry Guy to reuse your unnecessarily terse comment, it is you who are wrong.
>
>Multiphoton imaging clearly does limit the
>origins of any fluorescence but does not address
>the original problem, Poisson noise, about why
>confocal images appear noisy - lack of in focus
>photons, which you correctly pointed out.
>Deconvolution of widefield images does address
>Poisson noise since it efficiently uses all
>detectable photons and therefore is worthwhile.
>A deconvolved widefield image will often be
>preferable to a confocal or multiphoton image.
>There is also a semantic question about what
>constitutes noise and if unwanted out of focus
>photons can be returned to their origins then
>they cease to be noise and become signal.
>

Hi again,

Let's recognize that decon is at its base a
mathematical process relating to continuous,
mathematical functions. Here is works perfectly!
Therefore, that although the idea that "inversion
yields microscope truth" has appeal, it has
little to do with Fluorescence Microscopy,
especially that of living cells where the data is
discontinuous (pixellated) and where Poisson
noise is extremely important.

Maybe we need some numbers here. If we assume a
CCD read noise of +/- 5e/pixel, it will be
overshadowed by Poisson noise for any signal
level above 5x5= 25e/pixel. I submit that few
pixels in a widefield CCD data set record a
signal representing less than 25e (not
necessarily 25 counts in the memory but that
number multiplied by the gain-factor of the
camera to represent e/pixel). So Poisson Noise is
always dominant.

On the other hand, Gaussian noise is a lot easier
to model and, if there is a lot of out-of-focus
light (so that the in-plane contrast is say only
1% or even 10% of the total, then most pixels
have the almost the same number of electrons and
the square-root of these various numbers will
vary even less. This is the rationale for
assuming that Poisson Noise to be approximated by
a Gaussian without too much problem in widefield
decon.

As to "which is best":

I start by assuming that all 3D fluorescence
microscope data should be deconvolved.
Therefore, the question comes down to which is
the best method of data collection. Widefield
records some near-focus data that might be useful
and that confocal (may) exclude (depending on the
pinhole size). However, if there are bright
features farther from the focus plane, then they
will produce recorded photons that are probably
less useful. In the end it is all a matter of S/N
and this means statistics.

As a result. the decision depends on the geometry
of the specimen stain distribution and in
particular, the extent to which the greater QE of
the CCD (used in widefield. Greater QE reduces
Poisson Noise on the same light signal.),
compared to that of the PMT (used in confocal.
Depending on wavelength, the PMT is about 10x
worse than the CCD but getting better with the
new photon-counting, GaAsP detectors.), is offset
by the Poisson Noise introduced into the
widefield image by bright, out-of-focus features.
i.e., WF/Decon that does very well on
tissue-culture slides (at the UBC Course, for
instance) may have more problems, with embryos if
(and only if) they contain many stained features
in depth.)

Assuming that the acceptance angle of the
objective is 60 deg, the signal recorded in a
pixel of the size needed to Nyquist sample an
in-focus point object, will be about 80x lower
when the point object is only 1µm out of focus
than when it was in focus. This seems to indicate
that it will be hard to record any useful
(in-focus?) signal from features that are more
than 2 or 3 µm away from the focus plane.

However,  in WF, large, bright features much
farther from the focus plane will still produced
significant detectable signal (and its associated
Poisson Noise) and it is hard to see how this can
ever be thought useful.

Confocal and multiphoton will not record any signal from such features.

Personally, I feel that the "put the photons back
where they came from" wording is extremely
misleading.

Yes, we think that the processed 3D image
resembles the original object more closely than
does the raw data, but we seldom have any way to
check this. Essentially, the decon process is no
more immune to error than any other
extrapolation. As the data going into it noisy,
the output also has an error rate and the mere
fact that it looks convincing should not blind us
to the need to remember that its validity rests
on  assumptions that may not be valid.

For instance, decon assumes that the PSF is the
same over the whole field of view and at every
successive plane recorded. Neither of these
assumptions are "true" in any general sense (see
Handbook Chapters 11 and 20). An extreme example
is shown in Scanning, 24: 241-246 and also
Chapter 35. Here, the presence of the nucleus in
a living cheek cell not only greatly distorts the
PSF when recording features on its far side, it
also can displace them up to 6 micrometers. I
submit that there is no practical and general
method for "deconvolving" out this sort of
distortion.

In addition, 3D widefield data will include only
part of the light from emitted by features near
the edges (or even just outside) of the data
stack and hence this edge volume cannot be
deconvolved. The larger the PSF used, the larger
this volume of incomplete data will be.

And when time or photosensitivity constraints
limit one to collecting only 2D vs time data, the
confocal/multiphoton approach has much to offer,

So, can I suggest that we be content to be glad
that we have all these nice microscopes without
feeling that we have to win some sort of horse
race?

Also we need to we keep in mind that, as optimal
imaging conditions seldom overlap with optimal
cell viability, we need to remain cautious when
drawing conclusions from the high-resolution
aspects of any of the data that these techniques
produce.

Ciao,

JP

***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953                        
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 10-22, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/        Applications accepted after 11/15/12
               "If it ain't diffraction, it must be statistics." Anon.

>Quoting Guy Cox <[hidden email]>:
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>" The photons that are rejected by a pinhole usually come from
>>fluorophores within the specimen - deconvolution can be viewed as an
>>attempt to improve images by putting photons back to where they
>>probably originated, rather than to just reject them. Deconvolution
>>makes a more efficient use of the emitted photons.
>>
>>It is therefore possible to obtain an image by deconvolving a
>>widefield z series that, because of photobleaching and rejection of
>>photons by a pinhole, cannot be obtained from a confocal, even if
>>acquisition time was not the limiting constraint.
>>
>>Only in the narrowest sense, when only a single optical section is
>>required, is Guy correct in regarding out of focus fluorescence as
>>noise - perhaps signal of unwanted origin."
>>
>>WRONG!!  Yes, of course the photons come from
>>fluorophores within the specimen.  Where else
>>could they come from?  But they don't come from
>>where we are looking at and so they cannot be
>>assigned to the plane we are imaging.  They
>>belong in a different plane and should be
>>assigned there.  And in a confocal stack that
>>is exactly what will happen.  So of course
>>there is wasted fluorescence - and if we want
>>to avoid this the answer is rather simple - use
>>2-photon. Then there is NO out of plane
>>fluorescence.
>>
>>                                      Guy
>>
>>
>>Optical Imaging Techniques in Cell Biology
>>by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>>______________________________________________
>>Associate Professor Guy Cox, MA, DPhil(Oxon)
>>Australian Centre for Microscopy & Microanalysis,
>>Madsen Building F09, University of Sydney, NSW 2006
>>
>>Phone [phone]     Fax [phone]
>>              Mobile [phone]
>>______________________________________________
>>       http://www.guycox.net
>>
>>
>>
>>-----Original Message-----
>>From: Confocal Microscopy List
>>[mailto:[hidden email]] On
>>Behalf Of Jeremy Adler
>>Sent: Monday, 31 October 2011 11:08 PM
>>To: [hidden email]
>>Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>
>>
>>
>>Quoting Guy Cox <[hidden email]>:
>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>Daniel White wrote:
>>>
>>>" in a confocal you throw away most of the signal, as its out of focus.
>>>So as a result the images are often very noisy.  "
>>>
>>>This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>>>noise, not signal.  If you have no SA (and, honestly, if you are
>>>seriously interested in high-resolution imaging that should be a given)
>>>then a confocal microscope with the pinhole set at 1 Airy diameter
>>>throws away no signal at all.  So why are confocal images often noisy?
>>>Well, it's just statistics.  If you take a wide-field image with a 1
>>>second exposure each point is exposed for one second.  If you take a
>>>confocal image at 512 x 512 for 1 second then each point is exposed for
>>>~4 microseconds.  The difference is rather substantial ...
>>>
>>>                                            Guy
>>>
>>>Optical Imaging Techniques in Cell Biology
>>>by Guy Cox    CRC Press / Taylor & Francis
>>>      http://www.guycox.com/optical.htm
>>>______________________________________________
>>>Associate Professor Guy Cox, MA, DPhil(Oxon)
>>>Australian Centre for Microscopy & Microanalysis,
>>>Madsen Building F09, University of Sydney, NSW 2006
>>>
>>>Phone [phone]     Fax [phone]
>>>              Mobile [phone]
>>>______________________________________________
>>>       http://www.guycox.net
>>>
>>>
>>>-----Original Message-----
>>>From: Confocal Microscopy List [mailto:[hidden email]]
>>>On Behalf Of daniel white
>>>Sent: Monday, 31 October 2011 8:30 PM
>>>To: [hidden email]
>>>Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>Hi Peter,
>>>
>>>On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>>>wrote:
>>>
>>>>
>>>>Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>>>From:    Peter Werner <[hidden email]>
>>>>Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>>
>>>>An interesting point was made here by Jim Pawley:
>>>>
>>>>>I agree that sampling a bit higher than Nyquist never hurts,
>>>>>especially if you deconvolve (as you always should), but I think
>>>>>that it is a mistake to think that one can "separate" out the noise
>>>>>by decon. I think that noise is pretty fundamental.
>>>>
>>>>I had always heard that if you're doing confocal microscopy, at least
>>>
>>>>point-scanning confocal with a pinhole size of 1AU or smaller, that
>>>>deconvolution was superfluous, because you shouldn't be getting out of
>>>
>>>>focus light. So what is gained by deconvolution when one is sampling
>>>>voxel by voxel?
>>>
>>>in a confocal you throw away most of the signal, as its out of focus.
>>>So as a result the images are often very noisy.
>>>Good contrast.... but high Poisson distributed photon shot noise
>>>from only measuring a handful of photons.
>>>
>>>So usually one needs to do something about that noise...
>>>we want to separate the real signal from the noise.
>>>
>>>Often a Gaussian or mean filter is applied... which suppresses the noise
>>>
>>>by smoothing it out... but it also smooths the real signal, so
>>>effectively you lose
>>>the contrast and resolution that was the whole point of doing confocal.
>>>
>>>The smart way to suppress the noise, but keep the contrast and
>>>resolution
>>>is to do deconvolution.
>>>Deconvolution using a max likelyhood method uses the known shape of the
>>>PSF
>>>to make a best guess model of the real fluorophore distribution in the
>>>sample.
>>>You tell the deconvolution algorithm how noisy the image is (you have to
>>>guess
>>>unless you take 2 images and measure it)
>>>then it attempts to throw out the noise and keep the real signal,
>>>resolution and contrast intact.
>>>
>>>D
>>>
>>>>
>>>>Peter G. Werner
>>>>Merritt College Microscopy Program
>>>
>>>Dr. Daniel James White BSc. (Hons.) PhD
>>>
>>>Leader - Image Processing Facility,
>>>Senior Microscopist,
>>>Light Microscopy Facility.
>>>
>>>Max Planck Institute of Molecular Cell Biology and Genetics
>>>Pfotenhauerstrasse 108
>>>01307 DRESDEN
>>>Germany
>>>
>>>[phone] (German Mobile)
>>>[phone] (Work phone at MPI-CBG)
>>>[phone] (Fax MPI-CBG LMF)
>>>chalkie666 Skype
>>>http://www.bioimagexd.net        BioImageXD
>>>http://fiji.sc                                        Fiji -  is just ImageJ
>>>(Batteries Included)
>>>http://www.chalkie.org.uk                Dan's Homepages
>>>https://ifn.mpi-cbg.de                        Biopolis Dresden Imaging
>>>Platform (BioDIP)
>>>dan (at) chalkie.org.uk
>>>( white (at) mpi-cbg.de )
>>>
>>>-----
>>>No virus found in this message.
>>>Checked by AVG - www.avg.com
>>>Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>>
>>
>>
>>
>>Jeremy Adler
>>IGP
>>Rudbeckslaboratoriet
>>Daghammersköljdsväg 20
>>751 85 Uppsala
>>Sweden
>>
>>0046 (0)18 471 4607
>>
>>-----
>>No virus found in this message.
>>Checked by AVG - www.avg.com
>>Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>-----
>>No virus found in this message.
>>Checked by AVG - www.avg.com
>>Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11
>>
>
>
>
>Jeremy Adler
>IGP
>Rudbeckslaboratoriet
>Daghammersköljdsväg 20
>751 85 Uppsala
>Sweden
>
>0046 (0)18 471 4607

--
***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953                        
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 10-22, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/        Applications accepted after 11/15/12
               "If it ain't diffraction, it must be statistics." Anon.

-----
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[Vitaly Boyko]

I am looking for a script (plugin) for quantitative analys
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear All,

I am looking for a script (plugin) for quantitative analysis of dymanic behavior of Actin filaments (network) in 4D (xyzt) that would also include comparative analysis of two or more 4D image data sets? I am aware of MTrack2 plugin in Image J. I do believe that should be much more than that. 
Thank you. Vitaly 

[Lutz Schaefer]

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

Dear Guy,

just a few further comments:
Using out of focus light is of course only useful when within the axial
region of support of the PSF. In a high NA lens this will be around or less
than a micron. Further away, I agree with you - is for several reasons not
numerically feasible. For those who are interested, the reference to
Walter's paper showing the reconstruction of out of focus light is: W. A.
Carrington "Image restoration in 3d microscopy with limited data" SPIE Vol.
1205, Bioimaging and two dimensional spectroscopy (1990), p. 72ff.

You also write:
> ...from a bright point, and subtract it from
> that plane (that's deconvolution, or part of it).
>
Unless you are referring to Nearest Neighbour decon, which can not be
considered deconvolution to begin with as the model used is only an
approximate heuristic 2d - slice model, this statement is incorrect. The
inverse of the 3d forward problem never contains any subtractions. Instead,
the 're-assigned' contributions will sum up in a weighted fashion according
to the PSF in the in focus areas. Of course there is a more precise
mathematical explanation to all that. I want to refrain from that here, as
this information is available in the literature.

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: [phone]
Email:     [hidden email]
___________________________________

--------------------------------------------------
From: "Guy Cox" <[hidden email]>
Sent: Monday, October 31, 2011 19:57
To: <[hidden email]>
Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Jim,
>
> My comment was unnecessarily terse, and I apologise for that.  But it does
> get me cranky when people claim there's some value in the out of focus
> light.  You yourself pointed out that this is crazy, some time ago.  Just
> one micrometre from the focal plane the cone of light from a high NA lens
> is 6µm in diameter.  Go much further and it's everwhere!  (This is just
> repeating what you have said).   We can of course work out the intensity
> distribution expected 1µm from a bright point, and subtract it from that
> plane (that's deconvolution, or part of it).  But unless our sample is
> trivially sparse we can't predict the bright spot from its ghost at any
> substantial defocus.  (And if our sample is so sparse conventional
> resolution doesn't apply - that's STORM).  So we are not using all
> detectable photons in any meaningful sense.  What is more important, we
> are not using all excited fluorescence, since each fluorochrome molecule
> has a finite life.
>
> Confocal images will always be noisier than WF unless we scan for a very
> long time - we all seem to be agreed on that.  But both are exciting
> fluorescence outside the focal plane.  Multiphoton does not - we actually
> CAN make use of every detected photon.  We will need patience, granted,
> but if people are prepared to take hours acquiring a STORM or PALM dataset
> maybe they should think about taking several minutes to acquire a
> multiphoton one?  (And then, maybe, deconvolving it).
>
> (I fear that my µ symbols may not make it through the list server, so
> where you see  µm it means micrometres).
>
>                                      Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of James Pawley
> Sent: Tuesday, 1 November 2011 7:17 AM
> To: [hidden email]
> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>Sorry Guy to reuse your unnecessarily terse comment, it is you who are
>>wrong.
>>
>>Multiphoton imaging clearly does limit the
>>origins of any fluorescence but does not address
>>the original problem, Poisson noise, about why
>>confocal images appear noisy - lack of in focus
>>photons, which you correctly pointed out.
>>Deconvolution of widefield images does address
>>Poisson noise since it efficiently uses all
>>detectable photons and therefore is worthwhile.
>>A deconvolved widefield image will often be
>>preferable to a confocal or multiphoton image.
>>There is also a semantic question about what
>>constitutes noise and if unwanted out of focus
>>photons can be returned to their origins then
>>they cease to be noise and become signal.
>>
>
>
>
> Hi again,
>
> Let's recognize that decon is at its base a
> mathematical process relating to continuous,
> mathematical functions. Here is works perfectly!
> Therefore, that although the idea that "inversion
> yields microscope truth" has appeal, it has
> little to do with Fluorescence Microscopy,
> especially that of living cells where the data is
> discontinuous (pixellated) and where Poisson
> noise is extremely important.
>
> Maybe we need some numbers here. If we assume a
> CCD read noise of +/- 5e/pixel, it will be
> overshadowed by Poisson noise for any signal
> level above 5x5= 25e/pixel. I submit that few
> pixels in a widefield CCD data set record a
> signal representing less than 25e (not
> necessarily 25 counts in the memory but that
> number multiplied by the gain-factor of the
> camera to represent e/pixel). So Poisson Noise is
> always dominant.
>
> On the other hand, Gaussian noise is a lot easier
> to model and, if there is a lot of out-of-focus
> light (so that the in-plane contrast is say only
> 1% or even 10% of the total, then most pixels
> have the almost the same number of electrons and
> the square-root of these various numbers will
> vary even less. This is the rationale for
> assuming that Poisson Noise to be approximated by
> a Gaussian without too much problem in widefield
> decon.
>
> As to "which is best":
>
> I start by assuming that all 3D fluorescence
> microscope data should be deconvolved.
> Therefore, the question comes down to which is
> the best method of data collection. Widefield
> records some near-focus data that might be useful
> and that confocal (may) exclude (depending on the
> pinhole size). However, if there are bright
> features farther from the focus plane, then they
> will produce recorded photons that are probably
> less useful. In the end it is all a matter of S/N
> and this means statistics.
>
> As a result. the decision depends on the geometry
> of the specimen stain distribution and in
> particular, the extent to which the greater QE of
> the CCD (used in widefield. Greater QE reduces
> Poisson Noise on the same light signal.),
> compared to that of the PMT (used in confocal.
> Depending on wavelength, the PMT is about 10x
> worse than the CCD but getting better with the
> new photon-counting, GaAsP detectors.), is offset
> by the Poisson Noise introduced into the
> widefield image by bright, out-of-focus features.
> i.e., WF/Decon that does very well on
> tissue-culture slides (at the UBC Course, for
> instance) may have more problems, with embryos if
> (and only if) they contain many stained features
> in depth.)
>
> Assuming that the acceptance angle of the
> objective is 60 deg, the signal recorded in a
> pixel of the size needed to Nyquist sample an
> in-focus point object, will be about 80x lower
> when the point object is only 1µm out of focus
> than when it was in focus. This seems to indicate
> that it will be hard to record any useful
> (in-focus?) signal from features that are more
> than 2 or 3 µm away from the focus plane.
>
> However,  in WF, large, bright features much
> farther from the focus plane will still produced
> significant detectable signal (and its associated
> Poisson Noise) and it is hard to see how this can
> ever be thought useful.
>
> Confocal and multiphoton will not record any signal from such features.
>
> Personally, I feel that the "put the photons back
> where they came from" wording is extremely
> misleading.
>
> Yes, we think that the processed 3D image
> resembles the original object more closely than
> does the raw data, but we seldom have any way to
> check this. Essentially, the decon process is no
> more immune to error than any other
> extrapolation. As the data going into it noisy,
> the output also has an error rate and the mere
> fact that it looks convincing should not blind us
> to the need to remember that its validity rests
> on  assumptions that may not be valid.
>
> For instance, decon assumes that the PSF is the
> same over the whole field of view and at every
> successive plane recorded. Neither of these
> assumptions are "true" in any general sense (see
> Handbook Chapters 11 and 20). An extreme example
> is shown in Scanning, 24: 241-246 and also
> Chapter 35. Here, the presence of the nucleus in
> a living cheek cell not only greatly distorts the
> PSF when recording features on its far side, it
> also can displace them up to 6 micrometers. I
> submit that there is no practical and general
> method for "deconvolving" out this sort of
> distortion.
>
> In addition, 3D widefield data will include only
> part of the light from emitted by features near
> the edges (or even just outside) of the data
> stack and hence this edge volume cannot be
> deconvolved. The larger the PSF used, the larger
> this volume of incomplete data will be.
>
> And when time or photosensitivity constraints
> limit one to collecting only 2D vs time data, the
> confocal/multiphoton approach has much to offer,
>
> So, can I suggest that we be content to be glad
> that we have all these nice microscopes without
> feeling that we have to win some sort of horse
> race?
>
> Also we need to we keep in mind that, as optimal
> imaging conditions seldom overlap with optimal
> cell viability, we need to remain cautious when
> drawing conclusions from the high-resolution
> aspects of any of the data that these techniques
> produce.
>
> Ciao,
>
> JP
>
> ***************************************************************************
> Prof. James B. Pawley,
> Ph.  608-238-3953
> 21. N. Prospect Ave. Madison, WI 53726 USA
> [hidden email]
> 3D Microscopy of Living Cells Course, June 10-22, 2012, UBC, Vancouver
> Canada
> Info: http://www.3dcourse.ubc.ca/ Applications accepted after 11/15/12
>        "If it ain't diffraction, it must be statistics." Anon.
>
>>Quoting Guy Cox <[hidden email]>:
>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>" The photons that are rejected by a pinhole usually come from
>>>fluorophores within the specimen - deconvolution can be viewed as an
>>>attempt to improve images by putting photons back to where they
>>>probably originated, rather than to just reject them. Deconvolution
>>>makes a more efficient use of the emitted photons.
>>>
>>>It is therefore possible to obtain an image by deconvolving a
>>>widefield z series that, because of photobleaching and rejection of
>>>photons by a pinhole, cannot be obtained from a confocal, even if
>>>acquisition time was not the limiting constraint.
>>>
>>>Only in the narrowest sense, when only a single optical section is
>>>required, is Guy correct in regarding out of focus fluorescence as
>>>noise - perhaps signal of unwanted origin."
>>>
>>>WRONG!!  Yes, of course the photons come from
>>>fluorophores within the specimen.  Where else
>>>could they come from?  But they don't come from
>>>where we are looking at and so they cannot be
>>>assigned to the plane we are imaging.  They
>>>belong in a different plane and should be
>>>assigned there.  And in a confocal stack that
>>>is exactly what will happen.  So of course
>>>there is wasted fluorescence - and if we want
>>>to avoid this the answer is rather simple - use
>>>2-photon. Then there is NO out of plane
>>>fluorescence.
>>>
>>>                                      Guy
>>>
>>>
>>>Optical Imaging Techniques in Cell Biology
>>>by Guy Cox    CRC Press / Taylor & Francis
>>>      http://www.guycox.com/optical.htm
>>>______________________________________________
>>>Associate Professor Guy Cox, MA, DPhil(Oxon)
>>>Australian Centre for Microscopy & Microanalysis,
>>>Madsen Building F09, University of Sydney, NSW 2006
>>>
>>>Phone [phone]     Fax [phone]
>>>              Mobile [phone]
>>>______________________________________________
>>>       http://www.guycox.net
>>>
>>>
>>>
>>>-----Original Message-----
>>>From: Confocal Microscopy List
>>>[mailto:[hidden email]] On
>>>Behalf Of Jeremy Adler
>>>Sent: Monday, 31 October 2011 11:08 PM
>>>To: [hidden email]
>>>Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>>>
>>>*****
>>>To join, leave or search the confocal microscopy listserv, go to:
>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>*****
>>>
>>>
>>>
>>>
>>>Quoting Guy Cox <[hidden email]>:
>>>
>>>>*****
>>>>To join, leave or search the confocal microscopy listserv, go to:
>>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>*****
>>>>
>>>>Daniel White wrote:
>>>>
>>>>" in a confocal you throw away most of the signal, as its out of focus.
>>>>So as a result the images are often very noisy.  "
>>>>
>>>>This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
>>>>noise, not signal.  If you have no SA (and, honestly, if you are
>>>>seriously interested in high-resolution imaging that should be a given)
>>>>then a confocal microscope with the pinhole set at 1 Airy diameter
>>>>throws away no signal at all.  So why are confocal images often noisy?
>>>>Well, it's just statistics.  If you take a wide-field image with a 1
>>>>second exposure each point is exposed for one second.  If you take a
>>>>confocal image at 512 x 512 for 1 second then each point is exposed for
>>>>~4 microseconds.  The difference is rather substantial ...
>>>>
>>>>                                            Guy
>>>>
>>>>Optical Imaging Techniques in Cell Biology
>>>>by Guy Cox    CRC Press / Taylor & Francis
>>>>      http://www.guycox.com/optical.htm
>>>>______________________________________________
>>>>Associate Professor Guy Cox, MA, DPhil(Oxon)
>>>>Australian Centre for Microscopy & Microanalysis,
>>>>Madsen Building F09, University of Sydney, NSW 2006
>>>>
>>>>Phone [phone]     Fax [phone]
>>>>              Mobile [phone]
>>>>______________________________________________
>>>>       http://www.guycox.net
>>>>
>>>>
>>>>-----Original Message-----
>>>>From: Confocal Microscopy List [mailto:[hidden email]]
>>>>On Behalf Of daniel white
>>>>Sent: Monday, 31 October 2011 8:30 PM
>>>>To: [hidden email]
>>>>Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>>
>>>>*****
>>>>To join, leave or search the confocal microscopy listserv, go to:
>>>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>*****
>>>>
>>>>Hi Peter,
>>>>
>>>>On Oct 31, 2011, at 6:02 AM, CONFOCALMICROSCOPY automatic digest system
>>>>wrote:
>>>>
>>>>>
>>>>>Date:    Sun, 30 Oct 2011 13:09:10 -0700
>>>>>From:    Peter Werner <[hidden email]>
>>>>>Subject: Deconvolution of Confocal Images? (was: Airy Units)
>>>>>
>>>>>An interesting point was made here by Jim Pawley:
>>>>>
>>>>>>I agree that sampling a bit higher than Nyquist never hurts,
>>>>>>especially if you deconvolve (as you always should), but I think
>>>>>>that it is a mistake to think that one can "separate" out the noise
>>>>>>by decon. I think that noise is pretty fundamental.
>>>>>
>>>>>I had always heard that if you're doing confocal microscopy, at least
>>>>
>>>>>point-scanning confocal with a pinhole size of 1AU or smaller, that
>>>>>deconvolution was superfluous, because you shouldn't be getting out of
>>>>
>>>>>focus light. So what is gained by deconvolution when one is sampling
>>>>>voxel by voxel?
>>>>
>>>>in a confocal you throw away most of the signal, as its out of focus.
>>>>So as a result the images are often very noisy.
>>>>Good contrast.... but high Poisson distributed photon shot noise
>>>>from only measuring a handful of photons.
>>>>
>>>>So usually one needs to do something about that noise...
>>>>we want to separate the real signal from the noise.
>>>>
>>>>Often a Gaussian or mean filter is applied... which suppresses the noise
>>>>
>>>>by smoothing it out... but it also smooths the real signal, so
>>>>effectively you lose
>>>>the contrast and resolution that was the whole point of doing confocal.
>>>>
>>>>The smart way to suppress the noise, but keep the contrast and
>>>>resolution
>>>>is to do deconvolution.
>>>>Deconvolution using a max likelyhood method uses the known shape of the
>>>>PSF
>>>>to make a best guess model of the real fluorophore distribution in the
>>>>sample.
>>>>You tell the deconvolution algorithm how noisy the image is (you have to
>>>>guess
>>>>unless you take 2 images and measure it)
>>>>then it attempts to throw out the noise and keep the real signal,
>>>>resolution and contrast intact.
>>>>
>>>>D
>>>>
>>>>>
>>>>>Peter G. Werner
>>>>>Merritt College Microscopy Program
>>>>
>>>>Dr. Daniel James White BSc. (Hons.) PhD
>>>>
>>>>Leader - Image Processing Facility,
>>>>Senior Microscopist,
>>>>Light Microscopy Facility.
>>>>
>>>>Max Planck Institute of Molecular Cell Biology and Genetics
>>>>Pfotenhauerstrasse 108
>>>>01307 DRESDEN
>>>>Germany
>>>>
>>>>[phone] (German Mobile)
>>>>[phone] (Work phone at MPI-CBG)
>>>>[phone] (Fax MPI-CBG LMF)
>>>>chalkie666 Skype
>>>>http://www.bioimagexd.net BioImageXD
>>>>http://fiji.sc Fiji -  is just ImageJ
>>>>(Batteries Included)
>>>>http://www.chalkie.org.uk Dan's Homepages
>>>>https://ifn.mpi-cbg.de Biopolis Dresden Imaging
>>>>Platform (BioDIP)
>>>>dan (at) chalkie.org.uk
>>>>( white (at) mpi-cbg.de )
>>>>
>>>>-----
>>>>No virus found in this message.
>>>>Checked by AVG - www.avg.com
>>>>Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>>>
>>>
>>>
>>>
>>>Jeremy Adler
>>>IGP
>>>Rudbeckslaboratoriet
>>>Daghammersköljdsväg 20
>>>751 85 Uppsala
>>>Sweden
>>>
>>>0046 (0)18 471 4607
>>>
>>>-----
>>>No virus found in this message.
>>>Checked by AVG - www.avg.com
>>>Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
>>>-----
>>>No virus found in this message.
>>>Checked by AVG - www.avg.com
>>>Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11
>>>
>>
>>
>>
>>Jeremy Adler
>>IGP
>>Rudbeckslaboratoriet
>>Daghammersköljdsväg 20
>>751 85 Uppsala
>>Sweden
>>
>>0046 (0)18 471 4607
>
>
> --
> ***************************************************************************
> Prof. James B. Pawley,
> Ph.  608-238-3953
> 21. N. Prospect Ave. Madison, WI 53726 USA
> [hidden email]
> 3D Microscopy of Living Cells Course, June 10-22, 2012, UBC, Vancouver
> Canada
> Info: http://www.3dcourse.ubc.ca/ Applications accepted after 11/15/12
>        "If it ain't diffraction, it must be statistics." Anon.
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1411 / Virus Database: 2092/3985 - Release Date: 10/30/11
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1834 / Virus Database: 2092/4585 - Release Date: 10/30/11

[Daniel James White]

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

Hi Guy,

On Nov 1, 2011, at 6:04 AM, CONFOCALMICROSCOPY automatic digest system wrote:

> Date:    Mon, 31 Oct 2011 22:20:34 +1100
> From:    Guy Cox <[hidden email]>
> Subject: Re: Deconvolution of Confocal Images? (was: Airy Units)
>
> Daniel White wrote:
>
> " in a confocal you throw away most of the signal, as its out of focus.
> So as a result the images are often very noisy.  "
>
> This is often stated but IT IS TOTALLY UNTRUE.  What is out of focus is
> noise, not signal.

Its not really "noise" ... rather its out of focus "signal".
A confocal pinhole physically rejects most of that signal as its out of focus,
thats the idea of the confocal pinhole.

In widefield deconvolution microscopy we use that out of focus "signal"
and get better contrast and signal:noise( meaning shot noise mainly, but also background noise... not the same thing)

>  If you have no SA (and, honestly, if you are
> seriously interested in high-resolution imaging that should be a given)
> then a confocal microscope with the pinhole set at 1 Airy diameter
> throws away no signal at all.  

it does throw away out of focus signal... but thats what its supposed to do.
And it does still let through some amount of out of focus signal,
so its not perfect at excluding blur. There is still a PSF.

Where there is still blur there is a chance to deconvolve
(so long as the PSF is still bigger then 1 voxel)
and at the same time suppress the noise and increase the contrast.

So deconvolution of confocal images IS a good idea a large % of the time.

> So why are confocal images often noisy?
> Well, it's just statistics.  If you take a wide-field image with a 1
> second exposure each point is exposed for one second.  If you take a
> confocal image at 512 x 512 for 1 second then each point is exposed for
> ~4 microseconds.  The difference is rather substantial ...

Absolutely right Guy, its a huge difference. About 5 or 6 orders of magnitude in pixel dwell / exposure time.
Worse still, new modern PMTs are still not as sensitive as a good EMCCD camera..
so you lose even more signal.... so you have to put in much much much more light.

So here is the crunch:
People bang on about confocal being some kind of "Gold Standard"
for high resolution biological fluorescence imaging.

This is not true. There are different kinds of microscopes that are suited to different situations.
They all have pretty similar resolution (as defined by Abbe / Rayleigh / etc.)
but they vary greatly in contrast. Without good contrast you cant see the resolution thats there.

In short:
1) Confocal:
 Single point laser scanning confocal is slow for a large field of view but can be very fast for a very small field of view,
and required a large amount of energy for illuminating the sample per pixel as the pixel time is very short.
Spinning disc confocals get a  fast frame rate over a large field of view -  and illuminate the sample with less intensity
Much better for living stuff.

Single laser point scanning Confocal is really good for: Going really fast over a tiny field of view, and imaging difficult samples
where the scattering and optical propertied of the sample mean that widefield deconvolution wont work
because the PSF is not the same all over the sample, and/or scattering is a problem.

2) Widefield systems plus deconvolution:
can get away with much much less illumination light power and get a very high  
signal:noise and dynamic range compared to confocal systems, and be much faster for a large field of view
But you only get really good images
if the PSF is the same all over the sample, and you know the PSF shape, and there is little scattering.

3) Take home messages.
a)Fixed, dead, thin samples? Optically well behaved samples?
Widefield deconvolution is probably a good choice.
Confocal is not the best tool here.... it would be like driving a 300 million dollar military tank in a mortor bike grand prix race.
You will definitely finish the race, slowly... but you might not win... even though you have the most expensive "bike"

b) Difficult, thicker, scattery, optically naughty samples, in a small field of view?
Bring out the hammer - use the point scanning confocal or even 2 photon.
Otherwise, consider more efficient alternatives.

c) If the only tool you have is a hammer, every problem resembles a nail.
We have lots of tools and lots of different kinds of samples,
so we need to choose the right one,
and not always use point scanning confocal because someone misinformed said its the "Gold Standard"


Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666 Skype
http://www.bioimagexd.net  BioImageXD
http://fiji.sc                                        Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Biopolis Dresden Imaging Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Alberto Diaspro]

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Dear,
sorry for using this channel. In Genoa situation due to flood is terrible and today it is raining again. Unfortunately, six persons died, among them 1 baby and two girls (8 and 19 years old).
For all LAMBS friends, my self and family and LAMBS are ok. Let me be back soon with more appropriate news…sorry for this
All the best
Alby

[Zac Arrac Atelaz]

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My humble opinion is that you used the right channel, hope everything goes better Gabriel OH
 > Date: Tue, 8 Nov 2011 09:10:28 +0100

> From: [hidden email]
> Subject: Flood in Genoa
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear,
> sorry for using this channel. In Genoa situation due to flood is terrible and today it is raining again. Unfortunately, six persons died, among them 1 baby and two girls (8 and 19 years old).
> For all LAMBS friends, my self and family and LAMBS are ok. Let me be back soon with more appropriate news…sorry for this
> All the best
> Alby

     

[Konz, Richard]

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My best wishes to all those effected by the flooding in Genoa.

-Rich


Richard F. Konz, Jr.
Assistant Professor
Department of Medicine
Director, Flow Cytometry Core Facility
UMASS Medical School
55 Lake Avenue North
Room S5-322
Worcester, MA  01655
Office:  508-856-1598
Lab:  508-856-3276
Mobil:  508-868-6021
Lab Web Link:  http://www.umassmed.edu/facslab/index.aspx
My Faculty Page:  http://www.umassmed.edu/ivp/faculty/Konz.cfm
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Zac Arrac Atelaz [[hidden email]]
Sent: Tuesday, November 08, 2011 3:48 PM
To: [hidden email]
Subject: Re: Flood in Genoa

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My humble opinion is that you used the right channel, hope everything goes better Gabriel OH
 > Date: Tue, 8 Nov 2011 09:10:28 +0100

> From: [hidden email]
> Subject: Flood in Genoa
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear,
> sorry for using this channel. In Genoa situation due to flood is terrible and today it is raining again. Unfortunately, six persons died, among them 1 baby and two girls (8 and 19 years old).
> For all LAMBS friends, my self and family and LAMBS are ok. Let me be back soon with more appropriate news…sorry for this
> All the best
> Alby

[Tim Feinstein-2]

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Hello all,

We want to spec a four-laser launch for a new live cell system that will handle both CFP/YFP FRET and red/green imaging.   However, I am sad to see that gas lasers are no longer speccable and so the freebie 514 laser line is gone.  We would therefore have to spec a 514 DPSS and forego the far-red line.  

I was wondering whether there is a way to do more (or at least the same) with less.  488 nm excites YFP well enough, so in theory I could image CFP/YFP using a scan head dichroic with cutouts for 442 and 488 nm laser lines.  In my experience 442 nm laser excitation (via TIRF) causes negligible YFP excitation and 488 nm does not excite CFP, so it is possible that I could gain speed by passing everything through a single broad bandpass filter (e.g., 455-550 nm) and alternate excitations.   Assuming that cross-talk is not a problem, the most significant cost would be that I lose a decent chunk of CFP emission to the scan head dichroic, but in return I gain a 641 nm laser.  

Has anyone tried this?  Any feedback on or off-list would be much appreciated.  

Thanks and all the best,


TF  

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261