Re: Deconvolution of Confocal Images? (was: Airy Units)

Posted by Brian Northan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Confocal-Images-was-Airy-Units-tp6947651p6947704.html

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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:

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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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>
> [phone] (German Mobile)
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>