Posted by
Laevsky, Gary S. on
URL: http://confocal-microscopy-list.275.s1.nabble.com/SIM-redux-tp7580938p7580941.html
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Hi Michael,
The first thing I would ask is what does the background look like?
In the SIM reconstruction that I have seen in multiple platforms, there is an artifact that results in a "mottling" type of pattern. This is regular and uniform throughout the image. It can also be made to disappear depending on the parameters used for reconstruction.
The explanation I understand is quite basic, in relation to some of the knowledge held by people that monitor this list. And I'm sure they can expand, but it has to do with the combination of the different phases, during the reconstruction, used to capture the data.
The problem is, that it can be made to disappear by changing the reconstruction parameters. There is no "set" setting for the reconstruction. What I used to do is set my parameters so the background was uniform (if you stretch the heck out of the LUT (obviously not the live histogram), you can see the background better). Then I was confident that I was seeing "real" data.
If you see the pattern in the background, this may be the culprit. If the background is uniform, then George and Wendy's comments are more relevant.
Best,
Gary
Gary Laevsky, Ph.D.
Confocal Imaging Facility Manager
Dept. of Molecular Biology
Washington Rd.
Princeton University
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310
On Sep 7, 2013, at 4:58 PM, "Cammer, Michael" <
[hidden email]>
wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> We have a practical question about how we know whether a SIM result is showing real structure or not.
>
> We know from EM that the structure we are looking at is densely packed solid containing the protein of interest throughout, not hollow. However, the SIM result appears hollow. There is a possibility that the staining doesn't penetrate to the core, but we really don't know.
>
> Has anybody see this sort of problem with SIM where a structure known to be solid is reconstructed with a hollow core?
>
> Illustration at
http://www.flickr.com/photos/mcammer/9696746798/ or
https://nyumc.box.com/s/gyq3c3cw6p5aofb7gw84>
> We are using OMX Blaze with a 568 (or is it 561) nm laser.
>
> Thank you.
>
> _________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: (212) 263-3208 Cell: (914) 309-3270
>