paceTOMO seems to ignore tilt axis offset during its own realignToItem function

[Siyu-C-TOMO]

Dear Fabian,

Thanks for your amazing work and we found paceTOMO super helpful with our cryo-ET data collection pipeline. We tried to set up the newest version of paceTOMO and serialEM on Titan and encountered some issues during realignToItem.

Environment

• paceTOMO version: v1.9.2
• SerialEM version: SerialEM Version 4.2.1 64-bit, built May 19 2025
• Microscope / camera: Titan Krios G4 and Falcon 4
• OS: Windows

Summary
When using paceTOMO's realignToItem function in the new version, the acquired view magnification reference shows a fixed positional offset equal to the serialEM tilt axis offset. We had to manually move our target back to the original position when finalizing the targert, and the result appears to cancel the tilt-axis offset during collection and produces tilt-series with a large defocus slope. Using SerialEM's native realignToItem function does not produce this problem, but it is less robust since we rely more on the focusing area to align back all targets. We wonder whether this could be a bug in paceTOMO communicating with the updated version of serialEM, or is it more likely caused by our SerialEM configuration?

Best,
Siyu Chen

[eisfabian]

Dear Siyu,

Thank you for all the details!
It could indeed be a change in SerialEM that changed how the tilt axis offset is applied.
Can you confirm that when you set a tilt axis offset of 0, both realignToItem routines behave equivalently?
Do you have a Preview image to align to as well or just the View image?

Thanks and best wishes,
Fabian

[Siyu-C-TOMO]

Dear Fabian,

Thanks for the reply and the suggestion of this quick sanity check. I just tried to set the tlit axis offset to 0 and tried again. Indeed, the view image I took during target selection does not have any shift anymore and I don't need to right click and drag back to find the target. In this sense both realignToItem routines behave the same, and both will have defocus slopes.

We always follow the prompts and take preview images during target selection and use them to align the targets during a paceTOMO session. I also saw preview images being taken during paceTOMO initiation.

Thanks!
Siyu

[eisfabian]

Hi Siyu,

Thanks for the additional details!
2 things worth checking would be

• the "Center image shift on tilt axis" checkbox in the Image Alignment & Focus Panel
• the offset between View and Record after setting the tilt axis offset and before running the selectTarget script.

I would not expect the new SerialEM version to have made any changed to how this is handled, but I will check with some others if they have noticed anything similar.

Thanks again and best,
Fabian

[Siyu-C-TOMO]

Hi Fabian,

Thank you so much for helping us on this issue. I did another check and made sure the two points you mentioned are correct when I ran the script. If there could be any settings that may go wrong on SerialEM please let me know.

Best,
Siyu

[eisfabian]

Hi Siyu,

I will have to figure out if anything changed in regards to the tilt axis offset compared to previous SerialEM versions. I will see if I can do some tests in the coming weeks.
In the meantime, let me know if you figure out anything else!

Best,
Fabian