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

Sat May 24 05:23:45 PDT 2014

Dear Marin and Max,

our Cs corrected Titan initially had a linear magnification distortion of ~5% which is worse than the “normal” distortions one can typically observe for non-corrected microscopes. While this is not the primary use of a Cs corrector,  it is nevertheless possible to tune the corrector to minimize linear distortion. In our Cs corrected Titan we can reduce the linear distortion to <0.1% and these settings are stable over a very long period of time. Tuning the corrector for optimized linear distortion comes with a disadvantage of a slight increase in higher order aberrations. Those are, however, not resolution limiting in biological imaging and only of concern for material scientists. As Marin pointed out stable Cs corrector alignment requires a very stable high-tension and it takes indeed several hours (over night) to sufficiently stabilize after a restart.

In non-corrected microscopes linear distortion cannot be compensated and is dependent on the level of astigmatism in the microscope and therefore on the level of excitation of the stigmator which introduces the distortion. Since the basic level of astigmatism differs between microscopes one can expect the level of linear distortions to be in the range of 0.5-2%.

Best regards
Holger

Von: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] Im Auftrag von Max Haider
Gesendet: Montag, 19. Mai 2014 16:53
An: Marin van Heel
Cc: 3dem
Betreff: Re: [3dem] Magnification anisotropy at low mag settings on Titan Krios

Dear Marin,
I would like to correct your statement on the reasons of this observed distortion (a different magnification for two perpendicular orientations). As I could follow on the thread below this distortion depends on the magnification step and, therefore, it is definitely not caused within the Cs-corrector. When you change the magnification you only change the settings of the various lenses within the projector system and there is no change at all of any lens of the corrector. The point I made (or wanted to make) is that you need two stigmators at different locations in the column to modify this distortion by exciting one stigmator and to compensate it with a 2nd one at a different position. In order to compensate an existing distortion one has  to measure the power and orientation of the stigmators. There does not exist an automatic alignment tool because I guess there is a source of a 2-fold astigmatism somewhere in the projector and there is no stigmator nearby. This astigmatism can be observed and you compensate it with, for example, an objective lens stigmator leading to a distortion. Which varies with the magnification because you change the excitation of the lenses in the projector and, hence, you vary the force of the obj. stigmator within this area. As Zhiheng wrote FEI people are aware but currently there is no short time solution.
There are stigmators in the Cs-corrector but I am afraid those are too weak to be used for a 7% distortion and, again, one would have to use them for each magnification step with a different excitation. This I stated briefly to be used to circumvent or to reduce the distortion but neither these stigmators nor the Cs-corrector in general are causing the observed distortion.
Best regards
Max.

Yu, Zhiheng wrote:
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> [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.

Cheers,

Marin

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,

Marin

=======================================================

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.

     Thanks,

          Niko.

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