<div dir="ltr"><div>Hi again,</div><div><br></div>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.<div><br></div><div>Best,</div><div>Jonathan<br><div><br></div></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">Le jeu. 7 nov. 2024 à 10:10, Jonathan Bouvette <<a href="mailto:jonathan.bouvette@gmail.com">jonathan.bouvette@gmail.com</a>> a écrit :<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div dir="ltr">Hi Julien,<div><br></div><div>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.</div><div><br></div><div>Best,</div><div>Jonathan</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">Le jeu. 7 nov. 2024 à 10:00, Maufront Julien <<a href="mailto:julien.maufront@curie.fr" target="_blank">julien.maufront@curie.fr</a>> a écrit :<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div>
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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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Best regards,</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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<div id="m_-8023727315458892136m_-3816281068254384918divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>De :</b> Jonathan Bouvette <<a href="mailto:jonathan.bouvette@gmail.com" target="_blank">jonathan.bouvette@gmail.com</a>><br>
<b>Envoyé :</b> jeudi 7 novembre 2024 15:08<br>
<b>À :</b> Maufront Julien <<a href="mailto:julien.maufront@curie.fr" target="_blank">julien.maufront@curie.fr</a>><br>
<b>Cc :</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> 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">Le jeu. 7 nov. 2024 à 08:35, Maufront Julien <<a href="mailto:julien.maufront@curie.fr" target="_blank">julien.maufront@curie.fr</a>> a écrit :<br>
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<img size="87048" style="max-width: 910px;" src="cid:ii_1930727b601cb971f161"></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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<div id="m_-8023727315458892136m_-3816281068254384918x_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="m_-8023727315458892136m_-3816281068254384918x_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="m_-8023727315458892136m_-3816281068254384918x_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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