Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/SIM-redux-tp7580938p7580939.html
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Hi Michael,
What does SIMcheck tell you about your machine?
http://www2.bioch.ox.ac.uk/microngroup/software/SIMCheck.shtmlIf you are using fluorescent antibody detection, do you know the
antibodies penetrate all the way to the core (as you noted). If it
practical to use direct labeled fluorescent Fab instead of full length Abs?
What do GFP (or Venus, which should be brighter) fusion protein(s) show?
Are there any small molecules that find your favorite molecule? ... If
yes, are the small molecules fluorescent or could be conjugated to a
fluorophore? I'm thinking in particular of fluorescent-phalloidins and
fluorescent bungarotoxins as examples. A related method would be to
conjugate fluorophores to your protein of interest and (find a
microinjection setup and) microinject into your cells. Google: clare
waterman fluorescence speckle microscopy for where that could go (ex.
http://www.molbiolcell.org/content/22/21/3940.full ).
Does your Blaze also have TIRF/Monet mode?
... if yes, is the structure close enough to the coverglass to use it?
... if no, the OMX could be a great widefield scope to acquire data
(timelapse mode) for 3Bmicroscopy ...
http://www.coxphysics.com/3b/ ...
the ImageJ plugin is very slow, even on a small region of interest (so
far I've only played with it on their demo data set).
George
On 9/7/2013 3:58 PM, Cammer, Michael wrote:
> *****
> 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
>
>
>
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
http://works.bepress.com/gmcnamara/26/