http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Confocal-Images-was-Airy-Units-tp6947651p6951860.html
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.
approximate heuristic 2d - slice model, this statement is incorrect. The
inverse of the 3d forward problem never contains any subtractions. Instead,
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
Sen. Scientist
> *****
> 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 +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
>
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 +61 2 9351 3176 Fax +61 2 9351 7682
>>> Mobile 0413 281 861
>>>______________________________________________
>>>
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 +61 2 9351 3176 Fax +61 2 9351 7682
>>>> Mobile 0413 281 861
>>>>______________________________________________
>>>>
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
>>>>
>>>>+49 (0)15114966933 (German Mobile)
>>>>+49 (0)351 210 2627 (Work phone at MPI-CBG)
>>>>+49 (0)351 210 1078 (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
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>>>>
>>>
>>>
>>>
>>>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
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>>>No virus found in this message.
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>>>
>>
>>
>>
>>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
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> No virus found in this message.
> Checked by AVG - www.avg.com
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