Chapter 5 SerialEM

5.1. How to setup SerialEM to run different executable or at different kV?
5.2. When I collect montage with SerialEM, I can see clear boundary between pieces in overview, what's wrong?
5.3. Why I can not change camera setup parameters for Record, the options gray out?
5.4. How to collect polygon montage maps at multiple position?
5.5. There is beam with Focus parameter in Low Dose mode, but when I do Autofocus I get no beam, why?

5.1. How to setup SerialEM to run different executable or at different kV?

UPATE: With the later version of SerialEM, setting up it for different kV or for different set of property and calibration files to consult becomes even easier. Now it works with argument in "Target" area. For example, if there a folder called "80kV" existed under C:\Program Files\SerialEM, we can simply put "/80kV" in the as argument. This way, the setting file can be shared and doesn't need to be edited.


C:\Program Files\SerialEM\Develop\SerialEM.exe /80kV

To run a different executable, e.g., develop version or release version, go through following steps.

  1. Right click on the SerialEM shortcut icon on your desktop and select "Properties".

  2. For develop version, fill in "Target" area with

    
C:\Program Files\SerialEM\Develop\SerialEM.exe
    
  3. Or for stable release version, fill in "Target" area with

    
C:\Program Files\SerialEM\SerialEM.exe
    

To run SerialEM at a different kV, say 80, do the following steps.

  1. Make sure a new folder exist, if not, make one. Here is an example.

    
Z:\xuchen\SerialEM-settings\80kV
    
  2. Right click on SerialEM shortcut, select "Property" and put the above folder path into "Start in" area.

    
Z:\xuchen\SerialEM-settings\80kV 
    
  3. Start SerialEM and exit. This will create a new setting file in the above folder.

  4. Edit the newly generated setting file SerialEMSettings.txt in above folder, and modify the "System Path" entry to reflect where the Property and Calibration files locate. In this case of 80kV, input the line.

    
SystemPath      C:\Program Files\SerialEM\80kV
    

5.2. When I collect montage with SerialEM, I can see clear boundary between pieces in overview, what's wrong?

This is due to intensity gradient across the beam. Usually, it is caused by gun tilt alignment being off. Performing gun alignment procedure should resolve this issue, you will have to re-take the gain reference file afterwards though.

5.3. Why I can not change camera setup parameters for Record, the options gray out?

You have a file opened and this file contains image(s) collected with Record parameter. For example, after acquiring low mag montage (LMM) map, the montage file is opened until you close it. The program expects new image taken with Record parameters will be appandable to the current stack file. This it is as designed, for convenience. You can either open a new file or close the current file. After that, Record parameters can be changed again.

5.4. How to collect polygon montage maps at multiple position?

Once you know, it makes perfect sense. But before that, it can be confusing and even frustrating.

This is a very common task. One finds multiple promising grid meshes and wants to collect polygon montage maps for all of them. Naturally, one would think to draw multiple polygons and then ask SerialEM to collect montage for each one of them. However, if you think about it, it makes sense to only draw one polygon and collect the same kind of polygon montage maps at each of the locations, e.g., the center of the good meshes picked, because most likely all the meshes will share the same or very similar orientation and dimensions. This is how SerialEM does it, very clever indeed.

The key point is that once a montage file is opened, it contains all the information needed to collect a montage map images, at current stage location. The only left task is to go to a location.

Lets take a look at the procedure for a single location.

  1. Define a polygon. This can be done by adding polygon from Navigator window. This is usually done at lower mag where entire mesh can be viewed, e.g. on a Low Mag Montage(LMM) map. Alternatively, you can also do it by (1) manually driving stage to each of the 4 corners of a single mesh and (2) adding current stage position and (3) define it as corner point. Then you can define the polygon from NavigatorMontage & GridsPolygon from Corners.

  2. While the polygon item is highlighted in Navigator window, NavigatorMontage & GridsSetup Polygon Montage.

  3. Respond to Montage Setup Window.

  4. Define the filename for montage stack. As soon as you click on "OK", the file is opened and the stage will move to the X, Y of the drawn polygon.

  5. However, you can but you don't have to collect on the drawn polygon location. If you move stage to any position, and then click "Start" button on Montage control panel, the montage map will be collected at this very last location.

With this in mind, collecting polygon montage at multiple location becomes very simple.

  1. Add point item on Navigator window and turn flag "A" on.

  2. Do the steps 1-4 as above for single point.

  3. NavigatorAcquire at Points ... → select Acquire Map Image as primary task and click "OK".

5.5. There is beam with Focus parameter in Low Dose mode, but when I do Autofocus I get no beam, why?

It is very likely that Objective Aperture blocks the beam. Autofocus uses tilt beam pairs. If the aperture size is too small, or if the set beam tilt angle is too large, e.g. 5 degree, then beam could get blocked when tilted. Usually, for 40 - 50 μm objective aperture, 1.6 degree tilt beam works fine.