http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Confocal-Images-was-Airy-Units-tp6947651p6947996.html
probably originated, rather than to just reject them. Deconvolution
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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 +61 2 9351 3176 Fax +61 2 9351 7682
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> -----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)
>
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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
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>
> +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
>
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>
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> (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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