Re: CONFOCALMICROSCOPY Digest - 6 Sep 2013 to 7 Sep 2013 (#2013-208)

[Wendy Salmon]

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Dear Michael,

My first suspicion is antibody exclusion.  It sounds similar to tubulin antibody staining at the midbody, a microtubule and protein structure that forms between two mammalian* cells undergoing cytokinesis (*maybe non-mammalian cells, too--I'm not a cytokinesis expert).  With antibody staining, there is a gap in the tubulin structure between the cells whereas fluorescently conjugated tubulin (either microinjected or conjugated to GFP) and EM show microtubules throughout the midbody.  (Antibody staining vs. EM: Figure 2a of http://www.sciencedirect.com/science/article/pii/S0962892412002048 ; microinjected rhodamine tubulin: http://jcb.rupress.org/content/144/2/305/F8.expansion.html).

I agree with George that the optimal option would be to generate samples that are not prone to antibody penetration problems.  

If that isn't possible, perhaps our favorite standard, fluorescent beads, could help?  Comparing images from beads that are stained throughout to images of beads stained only on the periphery should give you some confidence in whether there's a hardware problem.  Using beads with a similar size to your object of interest and similar fluorophore characteristics (brightness and photostability) would be best, of course.

As a side note related to George's suggested fluorophores: From my understanding photobleaching is a huge problem for SIM image reconstruction, so photostability is probably more important than brightness all else being equal.

Good luck!
Wendy

~~~~~~~~~~~~~~~~~~~~~~~
Wendy Salmon
Light Microscopy Specialist
Whitehead Institute for Biomedical Research
W.M. Keck Imaging Facility
9 Cambridge Center, Rm 447
Cambridge, MA 02142
c: 617-429-0158
e: [hidden email]
w: http://staffa.wi.mit.edu/microscopy/

----- Original Message -----
From: "CONFOCALMICROSCOPY automatic digest system" <[hidden email]>
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Sent: Sunday, September 8, 2013 1:03:49 AM
Subject: CONFOCALMICROSCOPY Digest - 6 Sep 2013 to 7 Sep 2013 (#2013-208)

There are 2 messages totalling 119 lines in this issue.

Topics of the day:

  1. SIM redux (2)

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Date:    Sat, 7 Sep 2013 20:58:43 +0000
From:    "Cammer, Michael" <[hidden email]>
Subject: SIM redux

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We have a practical question about how we know whether a SIM result is show=
ing real structure or not.=0A=
=0A=
We know from EM that the structure we are looking at is densely packed soli=
d 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.=0A=
=0A=
Has anybody see this sort of problem with SIM where a structure known to be=
 solid is reconstructed with a hollow core?=0A=
=0A=
Illustration at http://www.flickr.com/photos/mcammer/9696746798/ or https:/=
/nyumc.box.com/s/gyq3c3cw6p5aofb7gw84=0A=
=0A=
We are using OMX Blaze with a 568 (or is it 561) nm laser.=0A=
=0A=
Thank you.=0A=
=0A=
_________________________________________=0A=
Michael Cammer, Assistant Research Scientist=0A=
Skirball Institute of Biomolecular Medicine=0A=
Lab: [phone]  Cell: [phone]=0A=
=0A=

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Date:    Sat, 7 Sep 2013 16:52:56 -0500
From:    George McNamara <[hidden email]>
Subject: Re: SIM redux

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Hi Michael,

What does SIMcheck tell you about your machine?
http://www2.bioch.ox.ac.uk/microngroup/software/SIMCheck.shtml

If 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: [phone]  Cell: [phone]
>
>
>    

--



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/

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End of CONFOCALMICROSCOPY Digest - 6 Sep 2013 to 7 Sep 2013 (#2013-208)
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