This tutorial shows how to define a randomly oriented matrix of holes and how to setup a multishot collection within this matrix. It is specific for the given grid, hole size and hole orientation and it needs to be therefore set up for each individual grid. However, the coordinates can be saved for the subsequent grid switching if you have enough time before the bake out.
Even though there is a dedicated function for both random and regular hole pattern within Serial EM, we have utilized a workflow based on a script written by Rouslan.
- Before you run the script, have a quick look on the grid and check the hole geometry (in other words look up when the nearest hole is going to be)
- Be sure to be in the center of square, this will give you enough space for shifting the image over multiple holes without hitting the grid bar
- Be sure to have a log file opened
- Go to the magnification used for image recording (typically 60k) and use the screen camera to center the image over a hole using Stage X,Y
- Assign ImageShift1 to knobs and run HolePatternDirections script
- This will remove the CL aperture, open the dialog (Fig. 1) and you will be advised to remove the slit. However, even though there is a smaller field of view, the features could be better visible with the filter (compare Fig 2a and 2b)
- Follow the instruction on the text dialog and center the the holes in five steps using Multi X,Y (image shift) and adjusting the beam shift (Beam Shift X, Y) if necessary
- The script is defining positions of individual holes and creating their pattern, you can select the amount of holes for the final adjustment (normally 3 is fine). Remember that whatever you select does not influence the shape of your pattern – that you can select later on.
- Script will define two vectors – REFINEDVECTOR1 and REFINEDVECTOR2 and will write them in the log (Fig. 4)
- Copy and paste these vectors to the Parameters script (see an example in Fig. 5 in red). If you want to keep the previous vectors, use # and be sure to mark the current grid ID.
- In order to select the exact hole pattern which will be used for the collection, you can choose one of the preselected (e.g. 3×3 pattern – Fig. 5 in blue) or you can design a new one by defining the x,y coordinates for every single hole. Be sure to keep all the other patterns as well using #.
- Your pattern for the given grid is now selected, you can further modify the script Parameters to get a proper Multishot setup.