<html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"></head><body style="overflow-wrap: break-word; -webkit-nbsp-mode: space; line-break: after-white-space;"><div style="display: block;">I agree with Steve. Here is the original article about stress relief. </div><div style="display: block;">This method drastically enhances the intactness of lamellas during TEM loading, therefore, increases numbers of tilt series taken from one grid.</div><div style="display: block;">My lamellas are usually about 12 microns in width. Mean error during Etomo reconstruction indicates it is also better to make not-so-wide lamellas for the quality of the tilt series.</div><div style="display: block;">Good luck!</div><div style="display: block;">Reika</div><div style="display: block;"><div style="-webkit-user-select: all; -webkit-user-drag: element; display: inline-block;" class="apple-rich-link" draggable="true" role="link" data-url="https://pubmed.ncbi.nlm.nih.gov/31536774/"><a style="border-radius:10px;font-family:-apple-system, Helvetica, Arial, sans-serif;display:block;-webkit-user-select:none;width:300px;user-select:none;-webkit-user-modify:read-only;user-modify:read-only;overflow:hidden;text-decoration:none;" class="lp-rich-link" rel="nofollow" href="https://urldefense.com/v3/__https://pubmed.ncbi.nlm.nih.gov/31536774/__;!!Mih3wA!GWh_LSiBtQGSydUZjFpuc1nd-akmzY0OXnmDji9USbHvdkPj2-5YHHq-km2qO98FOxRoAbL1Ekws_zmQlJzX8fiNMIPrCw$" dir="ltr" role="button" draggable="false" width="300"><table style="table-layout:fixed;border-collapse:collapse;width:300px;background-color:#E5E6E9;font-family:-apple-system, Helvetica, Arial, sans-serif;" class="lp-rich-link-emailBaseTable" cellpadding="0" cellspacing="0" border="0" width="300"><tbody><tr><td vertical-align="center" align="center"><img style="width:300px;filter:brightness(0.97);height:155px;" width="300" height="155" draggable="false" class="lp-rich-link-mediaImage" alt="pubmed-meta-image-v2.jpeg" src="cid:A5C915C9-6269-4033-86EA-A85E763BC951"></td></tr><tr><td vertical-align="center"><table bgcolor="#E5E6E9" cellpadding="0" cellspacing="0" width="300" style="font-family:-apple-system, Helvetica, Arial, sans-serif;table-layout:fixed;background-color:rgba(229, 230, 233, 1);" class="lp-rich-link-captionBar"><tbody><tr><td style="padding:8px 0px 8px 0px;" class="lp-rich-link-captionBar-textStackItem"><div style="max-width:100%;margin:0px 16px 0px 16px;overflow:hidden;" class="lp-rich-link-captionBar-textStack"><div style="word-wrap:break-word;font-weight:500;font-size:12px;overflow:hidden;text-overflow:ellipsis;text-align:left;" class="lp-rich-link-captionBar-textStack-topCaption-leading"><a rel="nofollow" href="https://urldefense.com/v3/__https://pubmed.ncbi.nlm.nih.gov/31536774/__;!!Mih3wA!GWh_LSiBtQGSydUZjFpuc1nd-akmzY0OXnmDji9USbHvdkPj2-5YHHq-km2qO98FOxRoAbL1Ekws_zmQlJzX8fiNMIPrCw$" style="text-decoration: none" draggable="false"><font color="#272727" style="color: rgba(0, 0, 0, 0.847059);">Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae - PubMed</font></a></div><div style="word-wrap:break-word;font-weight:400;font-size:11px;overflow:hidden;text-overflow:ellipsis;text-align:left;" class="lp-rich-link-captionBar-textStack-bottomCaption-leading"><a rel="nofollow" href="https://urldefense.com/v3/__https://pubmed.ncbi.nlm.nih.gov/31536774/__;!!Mih3wA!GWh_LSiBtQGSydUZjFpuc1nd-akmzY0OXnmDji9USbHvdkPj2-5YHHq-km2qO98FOxRoAbL1Ekws_zmQlJzX8fiNMIPrCw$" style="text-decoration: none" draggable="false"><font color="#808080" style="color: rgba(0, 0, 0, 0.498039);">nlm.nih.gov</font></a></div></div></td></tr></tbody></table></td></tr></tbody></table></a></div></div><div><br></div><div><br><blockquote type="cite"><div>On Jan 5, 2025, at 5:55 AM, Ludtke, Steven J. <sludtke@bcm.edu> wrote:</div><br class="Apple-interchange-newline"><div><meta charset="UTF-8"><span style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 13px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;">Ok, not to overstate the obvious, but in the images you're showing, I'd note a decided absence of milled strain reliefs? ie - minimally, parallel straight cuts adjacent and parallel to the lamella, or one of the more complicated jigsaw puzzle style approaches. There is a reason people make those...</span><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 13px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;"><br></div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 13px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;">Also a bit hard to get exact scale from your images. How wide are these lamella?<br><div><div><br><blockquote type="cite"><div>On Jan 5, 2025, at 1:39 AM, Long Gui <<a href="mailto:guilong@smart.org.cn">guilong@smart.org.cn</a>> wrote:</div><br class="Apple-interchange-newline"><div><font size="2"><b>***CAUTION:*** This email is not from a BCM Source. Only click links or open attachments you know are safe.</b></font><hr><div class="__aliyun_email_body_block"><div style="font-family: Tahoma, Arial, STHeitiSC-Light, SimSun;"><div style="clear: both;"><div style="margin: 0in; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; text-align: start; text-indent: 0px; word-spacing: 0px; white-space: normal; background-color: rgb(255, 255, 255);"><span style="color: rgb(36, 36, 36); font-style: normal; font-weight: 400; text-transform: none; font-family: helvetica; font-size: 14px;">Dear Colleagues,</span></div><div style="margin: 0in; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; text-align: start; text-indent: 0px; word-spacing: 0px; white-space: normal; background-color: rgb(255, 255, 255);"><span style="color: rgb(36, 36, 36); font-style: normal; font-weight: 400; text-transform: none; font-family: helvetica; font-size: 14px;"><br></span></div><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;">We are seeking your input regarding a recurring issue with broken or cracked lamella in our newly installed Titan Krios G4. Although lamella (200–300 nm thick) appear to be intact under the Aquilos, more than 60% of them show breakage or cracking after transfer into the Krios, making tilt-series data collection difficult. We have attempted multiple solutions—varying milling patterns, sputtering times, and testing different biological specimens—yet the problem persists.</span></div><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;"><br></span></div><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;">Additionally, we discovered that simply one round unloading/loading our lamella grids in the Krios Autoloader can break even previously intact lamella (Shown as the attached image). The engineer team insists that the Autoloader is functioning correctly. However, based on our previous experience with Titan Krios instruments, intact lamella from the Aquilos have typically exhibited a low chance of damage during loading.</span></div><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;"><br></span></div><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;">We would greatly appreciate hearing about your experiences:</span></div><ul style="padding: 0px 0px 0px 24px; margin: 2px 0px; list-style-type: disc;"><li><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;">How frequently do you observe lamella that are fine in the Aquilos but become damaged in the Krios?</span></div></li><li><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;">Do you have any strategies to test loading forces or reduce the rate of lamella breakage?</span><span style="font-family: helvetica;"></span></div></li></ul><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;"><br></span></div><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;">If you have encountered similar issues or have suggestions on mitigating lamella damage, we would welcome your advice and comments. Thank you for your time, and we look forward to hearing from you.</span></div><div style="clear: both;"><span style="font-family: helvetica; font-size: 14px;"><br></span></div><div><span style="font-family: helvetica; font-size: 14px;">Best Regards,</span></div><div><span style="font-family: helvetica; font-size: 14px;"><br></span></div><div><span style="font-family: helvetica; font-size: 14px;">Long Gui</span></div><p class="x_MsoNormal" data-olk-copy-source="MessageBody" style="margin: 0in; font-variant-ligatures: normal; font-variant-caps: normal; letter-spacing: normal; text-align: start; text-indent: 0px; word-spacing: 0px; white-space: normal; background-color: rgb(255, 255, 255);"></p><div style="clear: both;"><span style="font-size: 15px; font-family: Aptos, sans-serif; color: rgb(36, 36, 36); font-style: normal; font-weight: 400; text-transform: none;"><br></span></div><div><br class="webkit-block-placeholder"></div><p class="x_MsoNormal" data-olk-copy-source="MessageBody" style="margin: 0in; 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padding: 0px; border: 0px; outline: 0px; font-style: normal; font-weight: 400; text-transform: none; font-family: tahoma; font-size: 14px;">Shenzhen Medical Academy of Research and Translation (SMART)</span></div><div class="__aliyun_node_has_color" style="margin: 0px; padding: 0px; border: 0px; outline: 0px; font-family: Tahoma, Arial, STHeiti, SimSun; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; clear: both;"><span style="margin: 0px; padding: 0px; border: 0px; outline: 0px; font-family: tahoma; font-size: 14px;"><span class="__aliyun_node_has_color" style="margin: 0px; padding: 0px; border: 0px; outline: 0px; font-size: 14px; font-family: tahoma; font-style: normal; font-weight: 400; text-transform: none;">Email: guilong@smart.org.cn</span><span class="__aliyun_node_has_color" style="margin: 0px; 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