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

[Jonathan Bouvette]

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!Ft0GMrzF5GT9sei_DrUY143jROQFtDFzDfOC5pC22JczubuJjlZ68OOeseakUg4zlR_y0TItatw27TvjNBd8IZpQHIWVEA$ 
> <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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