[3dem] Issue with Thon rings

[Bill & Sue Tivol]

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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