Posted by
Daniel James White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Confocal-Images-was-Airy-Units-tp6947651.html
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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
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