http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Confocal-Images-was-Airy-Units-tp6947651p6948509.html
over and over, there are still misunderstandings. First of all,
forward imaging problem. This model almost never includes that out-of-focus
light is considered noise in a statistical sense. Instead it is considered
reassigns this light back to the focal plane, contrary to Guy's statement.
(estimate) known as maximum likelihood. Advanced deconvolution systems can
Sen. Scientist
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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."
>
> 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)
>
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>
>
>
>
> 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 )
>>
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> Jeremy Adler
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