[3dem] Broken/Cracked Lamella during loading to Titan Krios

[Reika Watanabe]

I agree with Steve. Here is the original article about stress relief. 
This method drastically enhances the intactness of lamellas during TEM loading, therefore, increases numbers of tilt series taken from one grid.
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.
Good luck!
Reika
https://urldefense.com/v3/__https://pubmed.ncbi.nlm.nih.gov/31536774/__;!!Mih3wA!GWh_LSiBtQGSydUZjFpuc1nd-akmzY0OXnmDji9USbHvdkPj2-5YHHq-km2qO98FOxRoAbL1Ekws_zmQlJzX8fiNMIPrCw$ 
Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae - PubMed
nlm.nih.gov


> On Jan 5, 2025, at 5:55 AM, Ludtke, Steven J. <sludtke at bcm.edu> wrote:
> 
> 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...
> 
> Also a bit hard to get exact scale from your images. How wide are these lamella?
> 
>> On Jan 5, 2025, at 1:39 AM, Long Gui <guilong at smart.org.cn <mailto:guilong at smart.org.cn>> wrote:
>> 
>> ***CAUTION:*** This email is not from a BCM Source. Only click links or open attachments you know are safe.
>> Dear Colleagues,
>> 
>> 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.
>> 
>> 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.
>> 
>> We would greatly appreciate hearing about your experiences:
>> How frequently do you observe lamella that are fine in the Aquilos but become damaged in the Krios?
>> Do you have any strategies to test loading forces or reduce the rate of lamella breakage?
>> 
>> 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.
>> 
>> Best Regards,
>> 
>> Long Gui
>> 
>> 
>> Junior Principal Investigator,
>> Institute of Bio-Architecture and Bio-Interaction,
>> Shenzhen Medical Academy of Research and Translation (SMART)
>> Email: guilong at smart.org.cn
>> Website: https://urldefense.com/v3/__https://smart.org.cn/en/research/investigator/content/post_1134771.html__;!!Mih3wA!GWh_LSiBtQGSydUZjFpuc1nd-akmzY0OXnmDji9USbHvdkPj2-5YHHq-km2qO98FOxRoAbL1Ekws_zmQlJzX8fgvhq85_g$  <https://urldefense.com/v3/__https://smart.org.cn/en/research/investigator/content/post_1134771.html__;!!Mih3wA!E7-528-k6WExFa9ifKq8D4_DPKgQclaIja2Ue38wnNsOp25FcZlFpxsiMtKOnJtmPeVY_WgBPe99e9Sj-Wsi$>
>> 
>> 
>> 
>> <Broken_lamella copy.jpg>_______________________________________________
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