[3dem] Magnification anisotropy at low mag settings on Titan Krios
Marin van Heel
marin.vanheel at googlemail.com
Fri Aug 22 09:03:58 PDT 2014
Dear All (and Holger, Max, and Niko),
I now realize that when astigmatism introduced at the level of the
objective lens is corrected much further down in the optical system then
that may result in anisotropic magnification of the imaging system…With
the Cs corrector in place (and with the objective lens stigmator
deactivated), astigmatism must be corrected by other stigmators
(quadrupole lenses) further downstream. The Cs corrector gives you ample
opportunities to correct the astigmatism but depending on how you do it
that can go at the cost of anisotropic magnification.The ~7%
magnification we found in one case, where this astigmatism correction
was apparently not performed correctly, was based on the image and the
amplitude spectrum attached. The images were collected with a Falcon-2
in movie mode (7 frames) and were aligned with the ALIGN-MOVIE command.
Interestingly the resulting spectrum shows systematic information in the
corners, at around 1.9A, that is, beyond the Nyquist frequency of
images. This grid hole obviously has a strong ice contamination.
Cheers
Marin
On 24/05/2014 09:23, Stark, Holger wrote:
>
> 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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>
>
> --
> ================================================================
>
> Prof Dr Ir Marin van Heel
>
> Professor of Cryo-EM Data Processing
>
> Leiden University
> NeCEN Building Room 05.27
> Einsteinweg 55
> 2333 CC Leiden
> The Netherlands
>
> Tel. NL: +31(0)715271424 // Mobile NL: +31(0)652736618
> Skype: Marin.van.Heel
> email: marin.vanheel(A_T)gmail.com
> and: mvh.office(A_T)gmail.com
>
> ----------------------------------------------
>
> Emeritus Professor of Structural Biology
>
> Imperial College London
> Faculty of Natural Sciences
> Biochemistry Building (Room 512)
> South Kensington Campus
> London SW7 2AZ, UK
> email: m.vanheel(A_T)ic.ac.uk
>
> Tel. UK: +44(0)2075945316 //Mobile: +44(0)7941540625
>
> ----------------------------------------------
> Visiting Professor at:
>
> Laborat�rio Nacional de Nanotecnologia - LNNano
> CNPEM/ABTLuS, Campinas, Brazil
> Brazilian mobile phone +55-19-99369051
>
> ------------------------------------------------------------------
>
> I receive many emails per day and, although I try,
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> -->
>
>
>
> --
> =======================================================================
> Dr. Max. Haider
> CEOS GmbH, Englerstr. 28, D-69126 Heidelberg
> Tel.: +49 (0)6221 89467 11, e-mail:Haider at CEOS-GmbH.de <mailto:Haider at CEOS-GmbH.de>
> FAX : +49 (0)6221 89467 29 mobile: +49 (0)170-2962663
--
================================================================
Prof Dr Ir Marin van Heel
Professor of Cryo-EM Data Processing
Leiden University
NeCEN Building Room 05.27
Einsteinweg 55
2333 CC Leiden
The Netherlands
Tel. NL: +31(0)715271424 // Mobile NL: +31(0)652736618
Skype: Marin.van.Heel
email: marin.vanheel(A_T)gmail.com
and: mvh.office(A_T)gmail.com
----------------------------------------------
Emeritus Professor of Structural Biology
Imperial College London
Faculty of Natural Sciences
Biochemistry Building (Room 512)
South Kensington Campus
London SW7 2AZ, UK
email: m.vanheel(A_T)ic.ac.uk
Tel. UK: +44(0)2075945316 //Mobile: +44(0)7941540625
----------------------------------------------
Visiting Professor at:
Laboratório Nacional de Nanotecnologia - LNNano
CNPEM/ABTLuS, Campinas, Brazil
Brazilian mobile phone +55-19-99369051
------------------------------------------------------------------
I receive many emails per day and, although I try,
there is no guarantee that I will actually read each incoming email.
Moreover, our Spam filters can be strikt and sometimes make
legitimate emails disappear (try the gmail accounts, alternatively)
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