SerialEM HowTo: Positioning Z

Chen Xu

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SerialEM is a powerful program for EM data collection. Although it is maybe originally designed for tomography data collection, it is equally powerful and flexible for single particle EM tasks.

For tomography data collection, it makes very good sense for specimen to be at eucentricity so that the image feature of the interest doesn't shift too far away when tilting the stage. In single particle application that doesn't involve tilting the specimen, to be at eucentricity is also a good idea. There are a couple of reasons for that. Microscope electronic optical performance would be optimal when specimen is at eucentricity. And if the specimen is always at eucentricity, then the optical condition for imaging at different date and time will be close enough that the data can be merged safely.

It is not hard to adjust the specimen to eucentricity after specimen rod insertion. However, the grid is usually not that flat, you can easily find the specimen is no longer at eucentricity level if moved to a new location that is a couple of meshes away. Moreover, even within the same mesh of a 400M gird, the specimen height can be very different from this side to the other. Therefore, for best data quality, it is required that specimen is to be adjusted to eucentricity at each record location. It is not easy to do that.

One of the manual ways to adjust to eucentricity is to adjust Z while wobbling the stage tilt until the image feature shift is minimal. Program like SerialEM can even do it automatically, by comparing the image feature disparity of tilt pairs. Both ways involve stage tilt. This introduces two problems. One is time consuming. The total time spent for eucentricity for all record locations will be significant. The other one is technical - for sided entry cryo holder like Gatan 626, tilting stage will inevitably cause bubbling again to the quieted LN2 surface in the dewar of the holder.

In this document, I want to show you how to use macros of SerialEM to go to eucentricity conveniently, without wobbling the stage.

You can also get pdf version of this document here.

Table of Contents
1 The Background
2 Standard Focus
3 Determine Eucentric Focus and Set to it
4 Calibrate Standard Focus
5 Adjust Z to Eucentric Height
6 Integrate Z_byG into Main LD Macro
7 Extra Note

1 The Background

On a TEM, three things are closely related and dependent on each other - Specimen Height, Focus and Beam Tilt Pivot-Points. Focus an image really means to focus an image of specimen at a particular height. For a specimen at a different height, one can still focus the image, but the Objective Lens will at different strength.

If a specimen is at Eucentric Height, it is also called at Eucentricity. And if image of it is focused, we call the objective lens at Eucentric Focus. Usually, we adjust beam tilt pivot-points relative to Eucentric Focus. This step is part of microscope alignment procedure. On a Tecnai TEM, we usually do it from Direct Alignment for a quick check.

After beam-tilt povit points (X,Y) are set at Eucentric Focus, if an image feature is not overlapped for + and - tilt beams, then it means either specimen is not at eucentric height or the objective lens is not at Eucentric Focus, or both. If we do know the specimen IS at Eucentric Height, we can then determine focus and adjust focus to any target level by studing the disparity of the same image feature under the image pairs of tilted beams. This is the method used by SerialEM and Leginon to adjust focus level. On the other hand, if we set the objective lens to Eucentric Focus and we can also estimate and adjust specimen to Eucentricity by utilizing the same image pair disparity. This is what I am going to show in this document. As you can see, this method doesn't involve any stage tilt.