Sporadic tracking issue at high tilts

[cyychong]

Hi everyone,

I face an issue where the PACEtomo seems to be mis-tracking at the higher tilts (we are currently using version 1.9.2c). It occurs sometimes (i.e. not all the time) but when it does, it happens for the whole dataset (i.e. all the lamellae I collect in that one dataset). So far the only fix I have found would be to reduce the groupSize back to 1, which doesn't make much sense to me. We typically use groupSize of 3.

I was wondering if anyone has faced a similar issue and could give some advice?

Thanks in advance!

[eisfabian]

Hi cyychong,

Thanks for reaching out!
I have not heard anyone report something like this.
Do all targets of a PACEtomo area lose tracking or just a subset?
Does it correlate with lamella thickness or contrast of features on the lamellae?

Best,
Fabian

[cyychong]

Hi Fabian,

Thanks for replying so quickly! All targets of one PACEtomo group lose tracking and it is always the same few tilts in one group. At my recent collection, this happened for all the groups. It always happens at the higher tilts and I can lose up to one third of my tilts from the mis-tracking.

I haven't seen any correlations with lamella thickness. I ran the same collection parameters on two different grids one week apart with lamellae of roughly the same thickness and it tracked well for one grid but mis-tracked for the other grid. Lamellae on both grids are fairly thin, mostly below 150 nm.

The other thing I normally do when selecting targets is to take the Preview image at a larger defocus of -10 microns to increase contrast. The data is collected at -4 microns defocus. Might the difference in defocus somehow affect the tracking (although I'm not sure why it would only affect at high tilts)?

Apologies for the long message. I appreciate you taking the time out to reply! Hope all is well with you.

Best,
Claris