Validating deconvolution

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sundar sundar
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Validating deconvolution

Hello all,

I'm using a deconvolution software to deconvolve a 3D stack. Does anyone know of any way in which I can measure the improvement over the original dataset? Are there any standard techniques for the same?

Thanks,
Sundar
Louis Villeneuve Louis Villeneuve
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RE Validating deconvolution


Compare raw and deconvolved  fluorescent beads (150-200nm)  in X-Y and mostly Z directions,

Louis




sundar <[hidden email]>@LISTS.UMN.EDU
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2009-09-21 10:08

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Validating deconvolution







Hello all,

I'm using a deconvolution software to deconvolve a 3D stack. Does anyone
know of any way in which I can measure the improvement over the original
dataset? Are there any standard techniques for the same?

Thanks,
Sundar
--
View this message in context: http://n2.nabble.com/Validating-deconvolution-tp3685530p3685530.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.



John Oreopoulos John Oreopoulos
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Re: Validating deconvolution

In reply to this post by sundar
Hi Sundar

I am unsure if there is a standard way to compare your "before" and  
"after" deconvolution image stacks, but one way that I've seen in the  
literature is to analyze identical line profiles in both image sets  
(either in the xy lateral dimensions or along the z-direction) and  
present the profiles on the same graph. If your deconvolution has  
worked, features of interest along your line profiles should be  
sharper in the deconvolved line. ImageJ has several standard  
functions and plugins that could be used to do this.

John Oreopoulos


On 21-Sep-09, at 10:08 AM, sundar wrote:

> Hello all,
>
> I'm using a deconvolution software to deconvolve a 3D stack. Does  
> anyone
> know of any way in which I can measure the improvement over the  
> original
> dataset? Are there any standard techniques for the same?
>
> Thanks,
> Sundar
> --
> View this message in context: http://n2.nabble.com/Validating- 
> deconvolution-tp3685530p3685530.html
> Sent from the Confocal Microscopy List mailing list archive at  
> Nabble.com.
Lutz Schaefer Lutz Schaefer
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Re: Validating deconvolution

In reply to this post by sundar
Sundar,

unfortunately, over human visual comparison there is no standard technique.
Adding to Louis suggestion, try to use larger fluorescent beads (1-5µm
depending on your objective) that are only stained on the surface. This way
you can see better how well aberrations had been dealt with.

Regards
Lutz

----- Original Message -----
From: "sundar" <[hidden email]>
To: <[hidden email]>
Sent: Monday, September 21, 2009 10:08 AM
Subject: Validating deconvolution


> Hello all,
>
> I'm using a deconvolution software to deconvolve a 3D stack. Does anyone
> know of any way in which I can measure the improvement over the original
> dataset? Are there any standard techniques for the same?
>
> Thanks,
> Sundar
> --
> View this message in context:
http://n2.nabble.com/Validating-deconvolution-tp3685530p3685530.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
Jose Viña Jose Viña
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Re: Validating deconvolution

In reply to this post by sundar
Hi Sundar,

> I'm using a deconvolution software to deconvolve a 3D stack. Does
> anyone know of any way in which I can measure the improvement over
> the original dataset? Are there any standard techniques for the same?

There are various kinds of 'improvement' deconvolution can bring you.
If you are interested in improving half intensity width (HIW) of
particles, best follow Louis' suggestion to measure HIW in lateral and
axial of beads. You will almost certainly learn interesting things
about your microscope.

If on the other hand you are working with widefield images and blur is
hampering your analysis, you might be only interested in reducing the
blur. Question is then how much reduction you need. A test object
could be a larger bead, but beads can have a high internal refractive
index, upsetting things.

Lastly, you might be working with noisy but well sampled data and
trying to measure object shapes. Then I would suggest to construct
synthetic objects and simulate the whole imaging, Poisson noise,
deconvolution and measurement chain, and see what the effect is on the
error in the measurement you plan to do.

Some links you might find useful:

A quantitative comparison of two restoration methods as applied to
confocal microscopy.
van Kempen GMP, van der Voort HTM and van Vliet LJ
http://www.svi.nl/g/?7028

A comparison of image restoration approaches applied to
three-dimensional confocal and wide-field fluorescence microscopy.
P. J. Verveer, M. J.  Gemkow and T. M.  Jovin.
http://www.svi.nl/g/?71a3

I hope this helps!

Cheers,

jose.
George McNamara George McNamara
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Re: Validating deconvolution

In reply to this post by sundar
sure, deconvolve a low NA objective lens data set and acquire a high
resolution confocal Z-series.

At 10:08 AM 9/21/2009, you wrote:

>Hello all,
>
>I'm using a deconvolution software to deconvolve a 3D stack. Does anyone
>know of any way in which I can measure the improvement over the original
>dataset? Are there any standard techniques for the same?
>
>Thanks,
>Sundar
>--
>View this message in context:
>http://n2.nabble.com/Validating-deconvolution-tp3685530p3685530.html