SerialEM Note: Work With A Hex Grid

Author

Chen Xu

Contact

<chen.xu@umassmed.edu>

Date Created

Nov. 22, 2024

Last Updated

Nov. 24, 2024

Abstract

Quantifoil released their new type of cryo grids - HexAuFoil. Both mesh shape and hole pattern arrangement in the meshes are hexagon. It has small holes and hole spacing. SerialEM has to develop some new features to deal with this.

Here I share some of our experience how to control hex grid using SerialEM.

Finding Holes for Hex grid

For hex, finding holes is similar to finding holes for a typical sqaure shaped hole pattern. But here you check the box next to “Hex grid”, as shown in red outline in the image below.

Fig.1 Hole Finder for Hex

_images/hole-finder-hex.png

Multiple Exposure Setup for Hex

And of course you need to setup multiple exposure accordingly.

Fig.2 Multiple Exposure for Hex

_images/multi-exposure-hex.png

In above image, in multiple exposure setup dialog, you need to check “Hex grid” here too. If you click on button [ Last Hole Vectors ] or [ Map Hole Vectors ], you will obtain Image Shift vectors that are the base to define a multiple exposure pattern. There is under-the-hood conversion from hole Stage geometry vectors to Image Shift vectors.

After finding holes, even you do not take any holes, the hole vectors become available. Those are the hole stage vectors. If it is an overview image of a map on which you use [ Map Hole Vectors ], it will not only convert them to image shift vectors (they are available in memory immediately), but also add two entries in nav file for that map item for hole vectors. So the map item will have this information in it. After nav file is saved, you can see the two entries for a map item as below.

...
HoleISXspacing = -0.264893 -0.995153 -0.73026
HoleISYspacing = 0.997737 0.284396 -0.713341
...

This would potentially help automation and for cases like geometry of holes having changes slightly from mesh to mesh and you have to obtain the image shift vectors dynamically.

Please note, the final image shift vectors used for data collection at high mag might be slightly different from freshly converted ones from MMM mag due to impefection by crossing such large mag distance. You should refine the final image shift vectors before committing massive collection.

The image shift vectors and how many rings for hexagon together define the actual multiple exposure pattern. Here how much of the maximum shift value for a pattern size should be considered.

Once the pattern is defined, you can easily combine it to all the holes found and result in a few point items of some holes. They are the centers of the multiple exposure patterns. The only task then is to get to those target centers.

Skipping a hole

The diameter of holes is 0.29 um, and spacing between closest holes is 0.6 um. While this provides some nice advantages for image quality over these small holes, it poses technical challenges for a scope that cannot give small enough beam while maintaining parallel beam condistions. On ThermoFisher Krios with 3 condenser lenses, one can easily have beam size about 0.6 - 0.7 microns, thus this is normally not a problem. However, for 200 keV scope with only 2 condenser lenses, the beam is usually too large - it will touch adjecent holes.

SerialEM provided a way to skip a hole.

After finding all the holes, just like before, the only thing one needs to do is to check “Use best 1/3 subset of holes”, as shown in red outline in the dialog window below.

Fig.3 Hole Finder for skipping a hole for Hex

_images/1:3-subset-of-holes-hex.png

This will only include 1/3 of the holes and still make them arranged in hexagon, although different from original all-hole hexagon. This is as if the other 2/3 holes are not there. It is pity to throw away 2/3 of good holes, but at least we can now collect on this hex grid with larger beam size.

It is then the same workflow to obtain multiple exposure pattern:

  • take the 1/3 holes found and save them into navigator

  • from multiple exposure setup dialog, click [ Last Hole Vectors ] or [ Map Hole Vectors ] to obtain base image shift vectors

  • define how many rings using dial buttons.

  • combine

Below is the pattern with 2/3 holes skipped. As you can see, larger beam size won’t cause issue anymore.

Fig.4 Multi Exposure pattern for skipping a hole

_images/1:3-subset-pattern-hex.png

After combining, the display for Aquire areas is show below. You can see 2/3 of holes skipped here.

Fig.5 Acquire Area Display for skipping a hole

_images/display-skip-hex.png

Erase Periodic Peaks

The compact, regular, sharp (high contrast) and small sized holes in an image of camera view can easily cause error for cross-correlation operation. This is because of periodic peals dominating the cross-correlation operation. Not only hex, the square shape UltrAuFoil grids also suffer from problem.

SerialEM has implemented a function to erasing priodic peaks for cross-correlation so real peaks get a chance to standout.

Here is an image showing cross-correlation peaks without erasing the periodic peaks. You can test this from menu Process - Show Cross-corr. The helpfile (https://bio3d.colorado.edu/SerialEM/betaHlp/html/menu_process.htm#hid_process_showcrosscorr) says:

Show Cross-corr command (Process menu)

Use this command to show the cross-correlation used to autoalign the image in buffer A
to the image in the autoalign buffer. The correlation will be placed into Buffer A and
existing images will be rolled to higher buffers, just as when a new image is acquired.

Fig.6 CC-peaks without erasing periodic peaks for Hex

_images/cc-peaks-without-erasing.png

After erasing the peaks, the domination is largely eliminated.

Fig.7 CC-peaks with erasing funtion turned on

_images/cc-peaks-with-erasing.png

To turn this function on,

  • For a task that involves cross-correlation such as eucentric rough and fine, autofocusing etc., check the box “Erasing Periodic Peaks in Align&Focus” in Image Alignement & Focus control panel.

  • For realign routine in LD mode, turn it on from menu Navigator - Erase Periodic Peaks in LD.

Warning

You should only use Erasing with AuFoil grids. Do not use it with normal carbon grids.