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

Thu Nov 7 07:15:42 PST 2024

Hi again,

I thought of another potential issue. We had a stage with a tilt axis
offset that was 3.2 um which made it behave a bit like what you see. Maybe
have Thermo check the tilt axis offset and the calibration. It could be
either off in the tomo5 calibrations (which i don't know how it works) or
if it's very off physically (>2 um), they may have to tweak your stage
slightly which is a simple procedure.

Best,
Jonathan

Le jeu. 7 nov. 2024 à 10:10, Jonathan Bouvette <jonathan.bouvette at gmail.com>
a écrit :

> 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 à 10:00, Maufront Julien <julien.maufront at curie.fr> a
> écrit :
>
>> 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>
>> a écrit :
>>
>>
>>
>> *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 <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!Byql3ZivXBmk6lqE9DPk8zahDbW2o6qgP4bFXJqLdtMSjcA88J1N1pEvO3i8Yikhgj7cDf_MQGsKHlqD2Kjo-Z-UTEFE-A$ 
>> <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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>> 3dem mailing list
>> 3dem at ncmir.ucsd.edu
>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>>
>>
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