[3dem] Magnification anisotropy at low mag settings on Titan Krios

Yu, Zhiheng yuz at janelia.hhmi.org
Mon May 19 07:22:31 PDT 2014

Hi Marin,

Thanks for following up on this thread and sharing the very useful feedback. The 2% or so magnification anisotropy/image distortion Niko referred to was from a Titan Krios without a Cs corrector. The main suspect is the projection system and FEI told us that they are still working on a solution.

Best Regards,
Zhiheng Yu
Manager of CryoEM Shared Facility, HHMI Janelia Farm Research Campus

From: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Marin van Heel
Sent: Monday, May 19, 2014 8:54 AM
Cc: 3dem; Max Haider
Subject: Re: [3dem] Magnification anisotropy at low mag settings on Titan Krios

Dear All,

I have spoken to Max Haider about the issue and he told me there is a straight-forward reason for this magnification behavior with the Cs corrector.  The combination of a quadrupole lens at the beginning of the corrector and another one at the end of the corrector can jointly cause this effect.  On our NeCEN Cs corrected instrument, we measured up to 7% magnification difference in two orthogonal directions in the first data sets we collected with a new Falcon-2 camera on our Cs-corrected instrument. This large difference in magnification has since been corrected by re-adjusting the corrector but this issue remains a concern for high resolution work. In particular, Rishi and Sacha found that the fine tuning of the Cs Corrector is only effective/stable after the high tension has been on and constant for some 10-12 hours.



On 04/04/2014 08:10, Marin van Heel wrote:

Dear All,

This could also be due to astigmatism in the illumination system. Such astigmatism would mean that the illumination is not parallel to the optical axis in (at least) one direction leading to anisotropic magnification effects as per our paper:

G. van Duinen,  M. van Heel, and A. Patwardhan, Magnification variations due to illumination curvature and object defocus in transmission electron microscopy, Opt. Express 13 (2005) 9085-9093.
Hope this helps,



On 04/04/2014 02:17, Nikolaus Grigorieff wrote:

Dear Colleagues,

We have recently noticed a problem with anisotropic magnification on one

of our Titan Krios microscopes. When recording data at a nominal

magnification of 29,000x, there seems to be an image distortion that

produces variable magnification in different directions of the image.

These variations were estimated using diffraction from gold particles to

be about 2%, a significant amount especially when working on large

assemblies such as viruses. The distortions can be approximately

corrected using image interpolation but this is not desirable, of

course. In one case, the resolution of a 700 Angstrom virus

reconstruction with data collected on a Gatan K2 direct electron

detector improved from 7 to 4 Angstrom after correcting for the distortions.

The severity of the distortion depends on the magnification setting. At

37000x magnification the magnification anisotropy is about 1% and

59,000x it appears to be undetectable. Since most Krios microscopes are

only calibrated for magnifications of 59,000x and higher, it is possible

that the problem we have observed also occurs on other instruments. This

will be particularly relevant for instruments that operate with the K2

detector mentioned above. The pixel size of this detector (5 microns)

usually demands magnifications settings of 29,000x and lower where the

distortions are significant. Users of detectors with a larger pixel size

(e.g. the Falcon direct electron detector) are less likely to experience

the distortions since they will typically use magnifications of 59,000x

and higher.

FEI have acknowledged the problem but at this point the cause is not

clear. We hope that the distortions can be corrected with a simple

recalibration of the projector lenses. We would be grateful if other

Titan Krios users could share their experience and possibly check if

distortions are detectable at lower magnification settings.




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