[3dem] Tomography, Multiposition Acquisition (Tomo5) - Defocus tracking fail

Thu Nov 7 07:49:19 PST 2024

Dear Julien,

Thank you for informing us about your case. We are sorry to hear that you are experiencing a defocus issue with the Off-axis Multishot acquisition using Tomography 5 Software.

As implied in your message, the most straightforward explanation for your observation is an incorrect attribution of angular conventions (tilt axis sign with respect to tilt axis assignment). We do compensate for defocus due to Z displacement correctly, given a certain convention. Our R&D team is aware of this problem in a number of systems and is actively seeking a more robust solution.

Please allow us to review your particular setup through direct communication. We apologize for the inconvenience.

Kind regards,
Reint

From Tomo, with Love ❤️

Reint Boer Iwema
Product Manager
Electron Microscopy

Thermo Fisher Scientific
Vlastimila Pecha 1282/12 | 627 00 Brno, Czech Republic
Mobile: +420 605 975 862
reint.boeriwema at thermofisher.com | thermofisher.com/tomography5

-----Original Message-----
From: 3dem <3dem-bounces at ncmir.ucsd.edu> On Behalf Of 3dem-request at ncmir.ucsd.edu
Sent: Thursday, November 7, 2024 16:10
To: 3dem at ncmir.ucsd.edu
Subject: 3dem Digest, Vol 207, Issue 8

CAUTION: This email originated from outside of Thermo Fisher Scientific. If you believe it to be suspicious, report using the Report Phish button in Outlook or send to SOC at thermofisher.com.

Send 3dem mailing list submissions to
        3dem at ncmir.ucsd.edu

To subscribe or unsubscribe via the World Wide Web, visit
        https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!HLrAl2XzZ3iCLg!CrHKvUG1Ggu8eG41OrE9YFtQbQlwW3Ttc4NGpmzk-LsdYg7pSr1OooWRYQ6dTX4WqS-_reVVfvsdseJZhYFY6PDM-FsJZ_E5qKssKQ$
or, via email, send a message with subject or body 'help' to
        3dem-request at ncmir.ucsd.edu

You can reach the person managing the list at
        3dem-owner at ncmir.ucsd.edu

When replying, please edit your Subject line so it is more specific than "Re: Contents of 3dem digest..."

Today's Topics:

   1. Re: Tomography, Multiposition Acquisition (Tomo5) - Defocus
      tracking fail (Jonathan Bouvette)

----------------------------------------------------------------------

Message: 1
Date: Thu, 7 Nov 2024 10:10:00 -0500
From: Jonathan Bouvette <jonathan.bouvette at gmail.com>
To: Maufront Julien <julien.maufront at curie.fr>
Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Tomography, Multiposition Acquisition (Tomo5) -
        Defocus tracking fail
Message-ID:
        <CAGqXUf0YZ49bgtjRKjiC2gZ6b2-1AgW2wSCzGHe94kEXBkf5dw at mail.gmail.com>
Content-Type: text/plain; charset="utf-8"

Hi Julien,

I have made a mistake in my calculation before (my calculator was in radians...). The predicted z offset is roughly sin(tiltangle)*distance from the tilt axis (stage y). You may calculate based on your coordinates for the tilt series you acquired. The other thing that may be happening is if your holder is not eucentric throughout the tilt series but usually we don't observe a very nice slope like yours in the defocus plot.

Best,
Jonathan

Le jeu. 7 nov. 2024 ? 10:00, Maufront Julien <julien.maufront at curie.fr> a ?crit :

> Hi Jonathan,
>
> Thank you for your quick answer.
>
> Actually I already sent a message to TFS like a month ago... In 
> principle it has been sent to "a specialist".
> But I am still waiting for feedback after repeated requests..
>
> You've been faster !
>
> Thank you and have a nice day.
>
> Best regards,
>
> *Julien Maufront*
> Research Engineer  ?  Cryo-Electron Microscopy Platform ______
>
> *Institut Curie  ? Research Center*
> *Physics of Cells and Cancer (UMR 168)* Molecular Microscopy of 
> Membranes
> *+33 1 56 24 67 82*
>
>
>
> ------------------------------
> *De :* Jonathan Bouvette <jonathan.bouvette at gmail.com> *Envoy? :* 
> jeudi 7 novembre 2024 15:08 *? :* Maufront Julien 
> <julien.maufront at curie.fr> *Cc :* 3dem at ncmir.ucsd.edu 
> <3dem at ncmir.ucsd.edu> *Objet :* Re: [3dem] Tomography, Multiposition 
> Acquisition (Tomo5) - Defocus tracking fail
>
> Hi Julien,
>
> This is something I observed in SerialEM before while doing off-axis 
> tomography. You are right that the defocus compensation is inverted 
> from that it should be. Tomo5 limits the image-shift distance to 5um, 
> at 60 degrees, without any correction, the defocus offset should be 
> 1.5um and you see about double of that indicating that the correction 
> is inverted. I would bring this up to TFS applications so that they 
> can fix the configurations for you as I wouldn't know how to do that 
> in Tomo5. I think you have enough data to support your case here.
>
> Best,
> Jonathan
>
> Le jeu. 7 nov. 2024 ? 08:35, Maufront Julien 
> <julien.maufront at curie.fr> a ?crit :
>
>
>
> *Julien Maufront*
> Research Engineer  ?  Cryo-Electron Microscopy Platform ______
>
> *Institut Curie  ? Research Center*
> *Physics of Cells and Cancer (UMR 168)* Molecular Microscopy of 
> Membranes
> *+33 1 56 24 67 82*
>
>
>
> ------------------------------
> *De :* Maufront Julien
> *Envoy? :* jeudi 7 novembre 2024 14:32 *? :* 3dem at ncmir.ucsd.edu 
> <3dem at ncmir.ucsd.edu> *Objet :* [3dem] Tomography, Multiposition 
> Acquisition (Tomo5) - Defocus tracking fail
>
> Dear 3dem community,
>
> During an application session on our Glacios with a ThermoFisher 
> Engineer we had the opportunity to try the multiposition acquisition 
> scheme '("*Off-axis Multi-shot acquisition*") within *Tomo5*.
> The software worked well for *tracking *positions, even far from the 
> tilt axis.
>
> But when I went to check the average *Defocus* of each tilt image 
> among the * full tilt series*, the result was disappointing.
>  I observed a huge *drift from the Target defocus* while going to 
> negative tilt angles or positive tilt angles.
> Here is an example of the measured defocus in regards of tilt angle 
> values within a Tilt Serie being acquired at off-axis position:
>
>
> So it seems that the z displacement of the off-axis position is not 
> well taken into account for target defocus compensation.
> Is it an expected behaviour ?
>
>
> On the other side I noted that the *tilt angles* registered (from -60 
> to
> +60) are inverted regarding the *global convention* (Cf:
> https://urldefense.com/v3/__https://wiki.dynamo.biozentrum.unibas.ch/w
> /index.php/Tilt_angle_convention__;!!Mih3wA!CxyOfV4yLyn6Yz23qX1HBVsVde
> denaesHeWlZ_GT6E4gRBpirWIhxAn1cwJHXyl-q-VU3jIVvpNrbMiGirBbrUPtoenuyQ$
> <https://urldefense.com/v3/__https:/wiki.dynamo.biozentrum.unibas.ch/w/index.php/Tilt_angle_convention__;!!HLrAl2XzZ3iCLg!EtUHtX4pBonU0MJaEHxIHwMRqPioLC-0rGu3X0vcdN0BN8CDvbS0Hb6WbucrNiGuxtVYE2m2vpfeKNAEXhHNbn_aVdbfAahNks3bVg$>).
> My tilt image being tagged -60 correspond to +60 according to the 
> convention and so on.
> Indeed when I tested the defocus from left side and right side of a 
> tilt image it shown an inconsistent behaviour in regards with the convention.
>
> In the meantime, the* tilt axis angle* registered in the mdoc files 
> show a suspicious negative sign: *-90.30*.
> If then we consider the tilt axis angle to be rather* +89.70* 
> (inverted orientation of the tilt axis), the registered tilt angles become consistent.
>
> So here I am wondering if the disappointing defocus behaviour during 
> off-axis tilt serie acquisition could be due to a wrong recorded 
> orientation of the tilt axis.
> Does it make sense ? Does anyone here had already faced such situation ?
>
>
> Thank you for your help.
>
>
> *Julien Maufront*
> Research Engineer  ?  Cryo-Electron Microscopy Platform ______
>
> *Institut Curie  ? Research Center*
> *Physics of Cells and Cancer (UMR 168)* Molecular Microscopy of 
> Membranes
> *+33 1 56 24 67 82*
>
>
>
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listin
> fo/3dem__;!!HLrAl2XzZ3iCLg!CrHKvUG1Ggu8eG41OrE9YFtQbQlwW3Ttc4NGpmzk-Ls
> dYg7pSr1OooWRYQ6dTX4WqS-_reVVfvsdseJZhYFY6PDM-FsJZ_E5qKssKQ$
>
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <https://urldefense.com/v3/__http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20241107/2282d8f3/attachment.html__;!!HLrAl2XzZ3iCLg!CrHKvUG1Ggu8eG41OrE9YFtQbQlwW3Ttc4NGpmzk-LsdYg7pSr1OooWRYQ6dTX4WqS-_reVVfvsdseJZhYFY6PDM-FsJZ_GRQzpJ6A$ >
-------------- next part --------------
A non-text attachment was scrubbed...
Name: image.png
Type: image/png
Size: 87048 bytes
Desc: not available
URL: <https://urldefense.com/v3/__http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20241107/2282d8f3/attachment.png__;!!HLrAl2XzZ3iCLg!CrHKvUG1Ggu8eG41OrE9YFtQbQlwW3Ttc4NGpmzk-LsdYg7pSr1OooWRYQ6dTX4WqS-_reVVfvsdseJZhYFY6PDM-FsJZ_E14FV2mA$ >

------------------------------

Subject: Digest Footer

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu
https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!HLrAl2XzZ3iCLg!CrHKvUG1Ggu8eG41OrE9YFtQbQlwW3Ttc4NGpmzk-LsdYg7pSr1OooWRYQ6dTX4WqS-_reVVfvsdseJZhYFY6PDM-FsJZ_E5qKssKQ$

------------------------------

End of 3dem Digest, Vol 207, Issue 8
************************************