[3dem] 'easy' (re)calibration of pixelsize?

[Wim Hagen]

Dear Tobias,

I tend to use Mag-I-Cal Si reflections. Especially on K2/K3 using superresolution, one can cover quite a large range of magnifications. I manually measure line profiles in FFT’s. For lower mags cross grating will do and if using SerialEM one can automatically acquire montages of cross gratings at higher mags, by binning things (say bin 4) this gives small enough image montages (see https://urldefense.proofpoint.com/v2/url?u=https-3A__bio3d.colorado.edu_SerialEM_betaHlp_html_setting-5Fup-5Fserialem.htm-23isMacro&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=CrknpSTnW7KrFuhfE_Dg6HSt2s3CFl-AW0TDKyQuFX4xHHuHsPWEEu6TQZbmXPG8&s=Zi3qD-PZ3zhezKOy563ZAP38Ww-qywefumqOAU4Yxnc&e= ). Another option is using Thallous Chloride (commercially available) as its spacings allow easier calibration of especially lower magnifications. Be aware of linear distortions when calibrating, on Titan those should have been corrected by TFS,. On other scopes this is not possible and one can go bad up to a few percent depending on the direction measured in Fourier space. See https://urldefense.proofpoint.com/v2/url?u=https-3A__arxiv.org_abs_1501.05928&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=CrknpSTnW7KrFuhfE_Dg6HSt2s3CFl-AW0TDKyQuFX4xHHuHsPWEEu6TQZbmXPG8&s=ZEhoNtQ4YQj3U_R1QYWjrtKcAX5La9XCXK5YAjS1xIk&e=  and https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ncbi.nlm.nih.gov_pmc_articles_PMC6760661_&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=CrknpSTnW7KrFuhfE_Dg6HSt2s3CFl-AW0TDKyQuFX4xHHuHsPWEEu6TQZbmXPG8&s=Ypy3QqzH0ESreaQfpSYUpTIIYHRV2yomFpTy_jktfsU&e=  for more info.
I have tried Dave Mitchell's free DifTools plugin for DM (https://urldefense.proofpoint.com/v2/url?u=http-3A__www.dmscripting.com_difftools.html&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=CrknpSTnW7KrFuhfE_Dg6HSt2s3CFl-AW0TDKyQuFX4xHHuHsPWEEu6TQZbmXPG8&s=o7ZArPcF1Vi8oySSUEJ-DKf_-iOaA-2AGINuPnnunGU&e= ) for the Mag-I-Cal images, sometimes it worked for me finding peaks in FFT's, but mostly not. Be aware that calibrating using the Cross Grating’s 463 nm repeat can be off a few percent, the metrology field made that clear to TEM vendors many years ago. In the end, for the typically used higher mags, I prefer a decent apoferritin grid, a handful of images, then let Relion or CRYOsparc give an accurate number assuming vendor specified Cs (see https://urldefense.proofpoint.com/v2/url?u=https-3A__relion.readthedocs.io_en_release-2D3.1_Reference_PixelSizeIssues.html&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=CrknpSTnW7KrFuhfE_Dg6HSt2s3CFl-AW0TDKyQuFX4xHHuHsPWEEu6TQZbmXPG8&s=oTqu9nRhxUoY-LWFW9T-NcpkugJ1ub7JlrmeROQgPEo&e= ) , although one probably still finds “noise" on pixel size numbers over time…. 
Please keep us posted if you find some useful reliable software to make this process easier.

Best,

Wim Hagen
EMBL Heidelberg

> On 3. Jan 2022, at 15:54, Tobias Furstenhaupt <furstenh at mpi-cbg.de> wrote:
> 
> Dear colleagues,
> 
> I would like to check/correct the pixelsize on our TEMs and am open for suggestions to make my life as easy as possible.
> For low mags I have the venerable crossgrating with a spacing of 463nm. My plan is to take images and use the cross-correlation function from DigitalMigrograph (DM) to get a precise spacing in pixels.
> For high and ultra-high mags I have the MAG*I*CAL that offers calibrated distances in the range of 10nm/100nm/1000nm/4um. Plan is to use Fiji or DM and do manual mesurements.
> 
> Does anybody have better suggestions, procedures or calibration standards that work well?
> 
> thanks alot in advance :)
> Tobias 
> 
> 
> ----------------------------------- 
> Tobias Fürstenhaupt, PhD 
> head of Electron Microscopy 
> Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) 
> Pfotenhauerstrasse 108 
> 01307 Dresden, Germany 
> 
> mail: furstenh at mpi-cbg.de 
> phone: (+49) (0)351 210-2690 
> cell: (+49) (0)176 / 44498706 (NEW!)
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