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Hi Jonathan,</div>
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Thank you for your quick answer.</div>
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Actually I already sent a message to TFS like a month ago... In principle it has been sent to "a specialist".</div>
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But I am still waiting for feedback after repeated requests.. </div>
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You've been faster !</div>
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Thank you and have a nice day.</div>
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<b>Julien Maufront</b></div>
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Research Engineer – Cryo-Electron Microscopy Platform</div>
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<b><i>Institut Curie – Research Center</i></b></div>
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<b>Physics of Cells and Cancer (UMR 168)</b></div>
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Molecular Microscopy of Membranes</div>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>De :</b> Jonathan Bouvette <jonathan.bouvette@gmail.com><br>
<b>Envoyé :</b> jeudi 7 novembre 2024 15:08<br>
<b>À :</b> Maufront Julien <julien.maufront@curie.fr><br>
<b>Cc :</b> 3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu><br>
<b>Objet :</b> Re: [3dem] Tomography, Multiposition Acquisition (Tomo5) - Defocus tracking fail</font>
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<div dir="ltr">Hi Julien,
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<div>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.</div>
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<div>Best,</div>
<div>Jonathan</div>
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<div dir="ltr" class="x_gmail_attr">Le jeu. 7 nov. 2024 à 08:35, Maufront Julien <<a href="mailto:julien.maufront@curie.fr">julien.maufront@curie.fr</a>> a écrit :<br>
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<img size="87048" style="max-width:910px" data-outlook-trace="F:1|T:1" src="cid:ii_19306f04b3fcb971f161"></div>
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<b>Julien Maufront</b></div>
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Research Engineer – Cryo-Electron Microscopy Platform</div>
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<b><i>Institut Curie – Research Center</i></b></div>
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<b>Physics of Cells and Cancer (UMR 168)</b></div>
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Molecular Microscopy of Membranes</div>
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<div id="x_m_2060735595630826574divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" color="#000000" style="font-size:11pt"><b>De :</b> Maufront Julien<br>
<b>Envoyé :</b> jeudi 7 novembre 2024 14:32<br>
<b>À :</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
<b>Objet :</b> [3dem] Tomography, Multiposition Acquisition (Tomo5) - Defocus tracking fail</font>
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Dear 3dem community,</div>
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During an application session on our Glacios with a ThermoFisher Engineer we had the opportunity to try the multiposition acquisition scheme '("<b>Off-axis Multi-shot acquisition</b>") within
<b>Tomo5</b>.</div>
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The software worked well for <b>tracking </b>positions, even far from the tilt axis.</div>
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But when I went to check the average <b>Defocus</b> of each tilt image among the <b>
full tilt series</b>, the result was disappointing.</div>
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I observed a huge <b>drift from the Target defocus</b> while going to negative tilt angles or positive tilt angles.</div>
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Here is an example of the measured defocus in regards of tilt angle values within a Tilt Serie being acquired at off-axis position:</div>
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<img id="x_m_2060735595630826574x_x_Image_x0020_2" width="481" height="270" style="width:361.49pt; height:203.24pt; max-width:1252px; min-width:auto; min-height:auto"></div>
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So it seems that the z displacement of the off-axis position is not well taken into account for target defocus compensation.</div>
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Is it an expected behaviour ?</div>
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On the other side I noted that the <b>tilt angles</b> registered (from -60 to +60) are inverted regarding the
<b>global convention</b> (Cf: <a href="https://urldefense.com/v3/__https:/wiki.dynamo.biozentrum.unibas.ch/w/index.php/Tilt_angle_convention__;!!HLrAl2XzZ3iCLg!EtUHtX4pBonU0MJaEHxIHwMRqPioLC-0rGu3X0vcdN0BN8CDvbS0Hb6WbucrNiGuxtVYE2m2vpfeKNAEXhHNbn_aVdbfAahNks3bVg$" id="x_m_2060735595630826574OWA0673481e-0848-830d-7f23-12a86688f055" rel="noopener noreferrer" target="_blank">
https://wiki.dynamo.biozentrum.unibas.ch/w/index.php/Tilt_angle_convention</a>). My tilt image being tagged -60 correspond to +60 according to the convention and so on.</div>
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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.</div>
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In the meantime, the<b> tilt axis angle</b> registered in the mdoc files show a suspicious negative sign: <b>-90.30</b>.</div>
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If then we consider the tilt axis angle to be rather<b> +89.70</b> (inverted orientation of the tilt axis), the registered tilt angles become consistent.</div>
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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.</div>
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Does it make sense ? Does anyone here had already faced such situation ?</div>
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Thank you for your help.</div>
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<b>Julien Maufront</b></div>
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Research Engineer – Cryo-Electron Microscopy Platform</div>
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<b><i>Institut Curie – Research Center</i></b></div>
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<b>Physics of Cells and Cancer (UMR 168)</b></div>
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Molecular Microscopy of Membranes</div>
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