Re: Deconvolution of 3D SIM data

Posted by Daniel James White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-3D-SIM-data-tp6251420p6260832.html

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Hi Andreas,

On Apr 9, 2011, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system wrote:

> Date:    Fri, 8 Apr 2011 11:48:46 -0400
> From:    Andreas Bruckbauer <[hidden email]>
> Subject: Re: Deconvolution of 3D SIM data
>
>
> When i generate a PSF for deconvolution using suitable beads, then image=
> the same beads again and deconvolve the image, i would expect to get real=
> ly tiny dots. See e.g. http://www.svi.nl/BeadsDeconvolutionExample=20

real tiny? The images of sub resolution objects dont get very much smaller after deconvolution...
what you really get is much higher contrast, so the features look "sharper"
You also get a little bit more resolution.. but thats not really the main point.
Its really about contrast.

> I would expect something similar for the OMX when the reconstruction is pe=
> rfect and includes proper deconvolution. However we get larger features,=
> still within the expected OMX resolution 120 nm in width and 300 nm in z,=
> but no dots.

No reconstruction or deconvolution will give images that contain features that are smaller
than the resolution limit of the system.

What do you mean by "dots" ?
Single 40 nm pixels?

In the OMX it's twice the conventional resolution,
so you cant get objects that appear smaller than 120 nm.
(actually... API say the OMX system sometimes seems to outperform theory slightly... but only a bit)

You should not an must not get single pixel object with the reconstructed pixel spacing of 40 nm (thats the spacing we have with the EM CCDs after 2x resolution increase from the physical 80 nm pixel spacing)

If you get smaller objects, its wrong, and an artifact. No?

> I think there is still some improvement possible either in=
> the setup of the instrument or the software.

Actually, in my hands it seems to do what theory predicts and no more.
There is no good reason to expect it to make smaller objects than 120 nm.

Certainly the careful alignment of the optics and the highest quality objective lens are critical,
as is the measurement of suitable SIM PSFs  and careful calibration/measurement of the parameters for the reconstruction...
the angles and the phases.

So, other than making it faster and more tolerant to sub optimal input data,
I dont think there is any improvement to be made in resolution.. so I dont see how you think there is room for resolution improvement?
Maybe you can share your ideas?

cheers

Dan


>
> best wishes
>
> Andreas
>
>
> =20
>
> =20

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
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Germany

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