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

Thu Nov 7 07:00:55 PST 2024

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>
Envoyé : jeudi 7 novembre 2024 15:08
À : 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 à 08:35, Maufront Julien <julien.maufront at curie.fr<mailto:julien.maufront at curie.fr>> a écrit :
[cid:ii_19306f04b3fcb971f161]

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
Envoyé : jeudi 7 novembre 2024 14:32
À : 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu> <3dem at ncmir.ucsd.edu<mailto: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:

[X]

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!C-18EHXclSwPzRMDQ3RoqxRynN3oyA5dgwqRmn4WyZaHLuOSZhdl3Ch-2qDJsxTYB2becGLm628is-XiRUgw8MjMCHecQA$ <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

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