Leginon

Your preset beam and image shift should be defined only when you are certain that you are at Eucentric height and relative to Eucentric focus by multiple confirmation such as doing the wabbling by hand. If that is true, Changing z-height position in order to reach Eucentric height at a particular position of a bent grid is equivalent to moving the stage up/down on a light microscope in order to get it focused with a fixed focus optical lens. Therefore, it should retain its alignment.

The adjustment you tried all seem correct although I don't have the complete information from your description. When you say that when it go through z-focus node, the z-height changed to 200 um, does this happened at the beginning of z focusing (that means the parent image was obtained at z-height of 200 um), or is it at the end (that means the focus sequence you choose measured a needed correction, and made the adjustment) ? If it is the latter case, and your recalibration and alignment did not make it better, please take the screen shots of the focus sequence settings for me or describe them in details. Also, check the log to find out how much it measured as the defocus at each step. If it is the former, you need to start making it a habit of do a rough eucentric height adjustment BEFORE grid atlas is taken.

In the case of one or more focus step in the sequence gives bad measurement, you should attempt to isolate it by switching on only one of them at a time and do the following simple test:

1. Manually put the stage to Eucentric height and reset defocus to eucentric focus at the magnification of the preset your active focus sequence use.
2. Check the autofocus result using Simulate Target Tool MSI_set-up_in_more_details Pay attention to the correlation peak position, especially and see if it matches the correlation map shown.

Because you have not seen this before, it is more likely your grid atlas was created when the grid was at 200 um z height.

One confusing point you mentioned is that you call this problem as a problem going from z-focus to drift monitor. In general, drift is not monitored in z-focus node because just a rough eucentric height adjustment is needed. Did you turn on drift monitoring at one of the focus step?

Anchi

Hi Anchi,

Thanks for your reply! First of all, yes, I had the drift monitoring turned on in the z-focus step. I turned it off, since it's unnecessary. Second, the grid I was shooting on, is pretty bend and z-height changes a lot going to different squares. I redid the grid atlas at eucentric height and it runs fine now. I appreciate your help!

Thanks,
Mandy

Good to know that this is a solvable case.

In the future, if you have a very bent grid and not wanting to recreate atlas for all the various height, you should consider trying to add a rough stage tilt based z-height measurement and correction as the first focus step in the Z Focus sequence. There is the inactive "Stage Wobble" in the default settings, you can just turn it on.

If that is not enough, add another one like in the instruction in Adding_a_new_focus_sequence_to_a_focus_node. If you use two stage wobbling, I recommend that you tilt at the same angle but use presets at different magnification to achieve a rough adjustment (lower mag, like sq preset) and then a fine adjustment (higher mag, like hl preset).