Re: SIM redux

Posted by Peng Xi-2 on Sep 10, 2013; 1:31pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/SIM-redux-tp7580938p7580945.html

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Dear Michael and Jim,
     We have imaged fluorescent nanodiamond with STED and SIM. The FNDs are
single molecule emitters with size of 35 nm diameter. STED has validated
that they are indeed in single particle. These particles are very good dots
for measuring the PSF of the imaging system.
     When we use SIM to image the particles, we found surprisingly that
even with 200nm out of focus, the reconstructed image shows hollow
structure, exactly as what you have seen. The hollow appears to be
insensitive to the parameters of reconstruction process.
     Based on the image you provided, I guess it may be from the fact that
your structure is slightly above the focal plane -- you may want to do a z
series of SIM to verify it.
     You can download the original data from:
http://bme.pku.edu.cn/~xipeng/FND-SIM.rar     Please pay special attention
to the last two frames.

Cheers,
Peng Xi
Ph. D.    Associate Professor
Dept. of Biomedical Engineering, College of Engineering
Peking University, Beijing, China
Tel: [phone]
Email: [hidden email]
http://bme.pku.edu.cn/~xipeng/




On Tue, Sep 10, 2013 at 3:19 AM, James Pawley <[hidden email]> wrote:

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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" <[hidden email]
>> >
>>  wrote:
>>
>>   *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<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/<http://www.flickr.com/photos/mcammer/9696746798/>or
>>> https://nyumc.box.com/s/**gyq3c3cw6p5aofb7gw84<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]
>>>
>>>
>
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