[3dem] 3dem Digest, Vol 129, Issue 23
Anchi Cheng
acheng at nysbc.org
Thu May 17 21:44:32 PDT 2018
Hi, Vishaka & Cindi,
I have heard of very high defocus problem after good autofocus measurement but both cases
were on Arctica and the source of the problem was failure in lens normalization. You should check
if lens normalization works properly.
There are simple ways to check if autofocus really failed. You need to use a specimen
with good contrast, put it at eucentric height and focus and watch the program goes through it.
If I recall correctly, EPU does not reset user defocus after autofocus but simply apply offset.
Therefore, it should be possible for you to check whether it indeed measured a bad value
by trackng the values you see in TUI.
As far as autofocus failure at near-in-focus, EPU, Leginon, SerialEM all apply defocus offset and
have ways to repeat the autofocus measurement at a different offset in case the
first attempt happens to be in-focus as Bill pointed out. If your problem is consistent, I would
rule this out as the reason.
Assuming that the software once worked, meaning that it was calibrated
with a good alignment, beam tilt (rotation center) alignment is what is normally required to
bring it back. Before you do so, check it again with specimen that gives you high
signal-to-noise ratio such as a grating replica. Other alignment that affects it are usually so stable
that it would be done by service engineer and hidden from users.
Most importantly, microscopes that are routinely used with automated data collection software
should not go off alignment easily. Therefore, big alignment adjustment such as a 20 um defocus
problem is more likely an indication of hardware or software setting (normalization settings) problem.
Hope this helps.
Anchi Cheng
> On May 17, 2018, at 8:21 PM, 3dem-request at ncmir.ucsd.edu wrote:
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> Today's Topics:
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> 1. Postdoc Position @ Case Western Reserve University (Jason Mears)
> 2. Re: Issue with Thon rings (Bill & Sue Tivol)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Thu, 17 May 2018 16:56:58 -0400
> From: Jason Mears <jason.mears at case.edu>
> To: 3dem at ncmir.ucsd.edu
> Subject: [3dem] Postdoc Position @ Case Western Reserve University
> Message-ID: <9E2C47CB-FBB1-4483-904D-EA57077328CB at case.edu>
> Content-Type: text/plain; charset=us-ascii
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> Post-doctoral position, Mears Group, Case Western Reserve University
>
> A postdoctoral research position is immediately available in the Mears Lab at Case Western Reserve University. Our goal is to study the structure and function of proteins involved in mitochondrial dynamics. Our research program integrates cryo-EM and biochemical approaches and the environment fosters interactions with other structural and mitochondrial biologists.
>
> Our EM facility is housed at the Cleveland Center for Membrane and Structural Biology (CCMSB). Users will have access to a JEOL 2200FSC equipped an Omega filter and a BioQuantum K3 camera, a TF20 equipped with a Teitz 4K camera, and a T12 with a 4K camera. Additional resources for specimen preparation and image processing are in place.
>
> A successful candidate will work collaboratively to define molecular interactions that drive membrane remodeling. Applicants should hold a PhD (or be within ~6 months of holding a PhD) in the area of biochemistry, biophysics, molecular biology or a related area. The ideal candidate will have experience in structural biology (cryo-electron microscopy or x-ray crystallography), computational biology, and/or data processing.
>
> Please send a brief statement of research interests and a curriculum vitae with references to jason.mears at case.edu
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> Jason Mears, Ph.D.
> Assistant Professor
> Department of Pharmacology
> Office: 216-368-3348
> Email: jason.mears at case.edu
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>
>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 17 May 2018 20:21:39 -0700
> From: Bill & Sue Tivol <wtivol at sbcglobal.net>
> To: "Schwartz, Cindi (NIH/NIAID) [E]" <cindi.schwartz at nih.gov>
> Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Issue with Thon rings
> Message-ID: <8EF39596-EF4E-4F93-9D2E-B247785D1719 at sbcglobal.net>
> Content-Type: text/plain; charset="utf-8"
>
> Hi Vishaka & Cindi,
> If the autofocus is set up to find focus, then shift to a user-defined defocus, you could end up at a high defocus without realizing it. Basically, for cryo specimens near focus, the image features are not strong compared to the noise, so autofocus sometimes gives the best cross-corellation on the noise, which can result is a random defocus. One test is to set the user-defined defocus at zero, then manually take repeated autofocus readings. The result of these should converge?usually by over-or under-shooting true focus, then oscillating about focus with a decreasing amplitude. I wrote a little article about this in Microscopy Today, and it applies to all the autofunctions I tested.
> Yours,
> Bill
>
>> On Mar 12, 2018, at 3:52 PM, Schwartz, Cindi (NIH/NIAID) [E] <cindi.schwartz at nih.gov> wrote:
>>
>> Hi Vishaka,
>>
>> Last week, we noticed a similar phenomenon on our newly installed Falcon III. We collected data with the Falcon III on our Krios using both EPU and manual collection via TIA. (Long exposures, many fractions, multiple magnifications) In our case, we saw that the thon rings weren?t missing per se, but we were getting extremely high defocus (~20 um defocus on some images) and so we couldn?t see the thon rings out to the edge. In the software, we have the focus set at -1 to -3. So we tried collecting manual images and got similar results (used EPU to switch states to ease imaging). We are positive we are at eucentric height and are at focus when we start. We checked using the diffraction pattern before imaging in nearby regions on the same ?flat? grid square.
>>
>> I wonder if there is a bug somewhere in the software/hardware because your image looks very underfocus. Could it be that instead of the normal defocus you think you should be getting, you are just getting some crazy defocused image?
>>
>> Are other people experiencing this? Is it something to do with the autofocus routine or the amount of signal in a focus area needed for cross correlation? Scope alignments?
>>
>> Thanks,
>> Cindi
>>
>>
>> --
>> Cindi L. Schwartz
>> Electron Microscopist
>> Rocky Mountain Labs/NIAID/NIH
>> 903 South 4th Street
>> Hamilton, MT 59840
>> 406-363-9228
>> Cindi.Schwartz at NIH.gov <mailto:Cindi.Schwartz at NIH.gov>
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>>
>> From: Vishaka Santosh [mailto:vs4p at virginia.edu <mailto:vs4p at virginia.edu>]
>> Sent: Friday, March 9, 2018 2:06 PM
>> To: 3dem at ncmir.ucsd.edu <mailto:3dem at ncmir.ucsd.edu>
>> Subject: [3dem] Issue with Thon rings
>>
>>
>> Hello everyone! I am having a rather bizarre issue when analyzing my data. It was collected at 96kC with an exposure of 3.01 seconds for 78 fractions. Using MotionCor2, I attempted to analyze the quality of the data and when I look at the Thon rings, they are either very weak or not present. Any one have any ideas what could be wrong? I didn't collect this on a phase plate and it was collected on a Titan Krios at 300kV
>> <image004.jpg><image005.jpg>
>>
>> --
>> Vishaka Santosh
>> UVA Class of 2011, BS in Chemistry with a specialization in Biochemistry
>> VCU Class of 2013, MS in Biochemistry
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