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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 <[log in to unmask]>:
> *****
> 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:[log in to unmask]] On Behalf Of Jeremy Adler
> Sent: Monday, 31 October 2011 11:08 PM
> To: [log in to unmask]
> 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 <[log in to unmask]>:
>
>> *****
>> 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:[log in to unmask]]
>> On Behalf Of daniel white
>> Sent: Monday, 31 October 2011 8:30 PM
>> To: [log in to unmask]
>> 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 <[log in to unmask]>
>>> 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
> IGP
> Rudbeckslaboratoriet
> Daghammersköljdsväg 20
> 751 85 Uppsala
> Sweden
>
> 0046 (0)18 471 4607
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Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden
0046 (0)18 471 4607
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