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Jim,
Check out this reference, from my friend and colleague Filip Braet
Imaging Fluorescently Labeled Complexes by Means of Multidimensional Correlative Light and Transmission Electron Microscopy: Practical Considerations
Kobayashi, K., Cheng, D., Huynh, M., Ratinac, K.R., Thordardsson, P., Braet, F.
2012
Methods in Cell Biology 111 , pp. 1-20
Guy
Guy Cox, Honorary Associate Professor
School of Medical Sciences
Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of James Pawley
Sent: Tuesday, 10 September 2013 5:20 AM
To: [log in to unmask]
Subject: Re: SIM redux
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Hello all,
This important discussion reminds me of what my post-doc tutor, Alan Boyde used to say "You can only recognize artifacts in microscopy to the extent that you can 'view' the same structure in a second (or
third...) way" (where "view" means "gain reliable structural information about")
This thread started when Michael Cammer told us about a structure that he had previously studied by (it turns out freeze-fracture, rotary shadow) TEM that looked different when viewed using SIM. i.e., he had followed the rule.
I would really like to hear responses from other listers who who have
1) viewed a structure in one of the enhanced LM methods and 2) looked at the same structure by another microscopical technique (SEM, TEM etc). I would be particularly interested to hear from those who noticed significant differences and also how they interpreted these differences (might specimen prep play a role.)
Cheers,
JP
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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"
><[log in to unmask]>
> 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
>>
--
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone 604-885-0840, email <[log in to unmask]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
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