Acquiring Multiple Tilt Series Automatically

Multiple tilt series can be run in a batch mode by using the Navigator to define positions where the series are to be taken, setting the parameters for the acquisition in advance as the positions are selected for tilt series in the Navigator, then starting the tilt series from the Navigator Acquire dialog box .

Points to consider: There are various considerations for using this capability:

• Maps. Each target position should be located within a medium-magnification Navigator map that is large enough to allow the position to be found reliably with the Realign to Item command. Such a map is referred to as an anchor map, and Navigator has features to simplify taking such maps. It is recommended that you also take a map at the magnification for the tilt series, and corresponding to a zero-degree view of the target area, as this map will allow more precise positioning, but this map is not essential.
• Beam centering.  You will probably want to use the beam autocentering task to protect against the beam position wandering during protracted data collection. To do so, you need to set up the task to work properly at the magnification where you are acquiring data, using the Beam Autocenter Setup dialog box . Once this is set up, you should test the task before data collection to see if its parameters need adjustment (run the Autocenter Beam command in the Tasks menu or use the Shift-E hot key).
• Cooking plastic sections.  If you are working with plastic sections, you can make the pre-exposure of the specimen be part of the batch operations. To do so, you need to set up the specimen cooking task using the Specimen Cooking Setup dialog box . Once this is set up, you can run that dialog to check the parameters before data collection and make sure that they are appropriate for the extent of area being acquired.
• Bidirectional series.  For reliable results, it may be necessary to take the tilt series in two directions from 0 tilt.  This will usually be the case with cryo-samples, where the walk-up to a high starting angle or tracking down from it may fail.  It may also be desirable to use a bidirectional series with other samples where there is difficulty focusing at high tilt, since it is generally easier to stay in focus as high tilt is approached in small steps from below than to establish good focus when starting the series at high tilt.
• Finding tilt range.  Often you need to set the tilt angle range separately for the individual tilt series, after assessing what range is feasible at each location. You may find the Tilt Series Range Finder helpful for assessing the tilt range. If you use it and then select angles at the end of the range in the Tilt Series Range Viewing dialog, then you need to open the relevant tilt series parameters (using the TS Parameters button in the Navigator) to have the selected angles become the limiting angles for that series.  Note that as of SerialEM 3.4.0, a batch tilt series is considered to be successful if an image that is too dim is encountered at a high enough angle, as specified by settings in the Tilt Series Batch Policies dialog. Thus, you may be able to skip assessing the ending angle.  Since the same criteria are applied to terminate the first part of a bidirectional series upon a dim image, you may be able to skip assessing the tilt range completely for such series.
• Eucentricity and Z height.  As always, the tilt series will run much more smoothly if the eucentric height is set for the specific location. Moreover, if the specimen is going to be pre-exposed, then the final eucentricity setting should be done after cooking. This can be achieved by selecting to refine eucentricity either in the Navigator Acquire dialog box or the Tilt Series Setup dialog. However, this routine cannot run reliably unless the Z height is within ~10 microns of eucentric. There are two options for taking care of this: select the Rough Eucentricity task in the Navigator Acquire dialog box, or set the eucentricity approximately by whatever means you prefer when you set up the series at each point. If you set the Z height after adding the Navigator item that is marked for a tilt series, select the item and change its Z height with the Update Z button in the Navigator.
• Error handling.  You need to make a basic choice of what should happen in case of an error: whether the tilt series should stop with a message or whether the Navigator should terminate it and go on to the next. The Tilt Series Batch Policies dialog has an option for this basic choice and some other options relevant to running tilt series or other automated operations. These include an option to have a separate log file for each series, and an option to control whether and when to close column/gun valves if a message box is open for a long time.
• Email messages.  Receiving email notification can be very helpful when running multiple series, especially if you select for a message box come up when there is an error. In such cases, an email will be sent before the box is opened. To send mail, the program needs to be able to contact an SMTP server and this server needs to be defined in the properties file. Once this has been configured, you will be able to enter an email address in the Tilt Series menu and select the option to have emails sent.
• Imaging states.  For each tilt series, you have the option to store an imaging state consisting of scope settings such as beam intensity (or, in Low Dose mode, the entire set of parameters for the record area), and Record parameters such as exposure time. In Low Dose mode, the axis position of the Focus area and an offset from center of a subarea selected in the Focus camera parameters is also stored.  This capability is obviously useful if you want a different state for some tilt series, or a different Focus subarea in Low Dose mode; it can also be used for the first tilt series to guarantee that a particular state is set then.  However, it need not be used to set different axis positions for Focus, because this is saved separately as part of the Navigator item.
• Saving to Navigator file.  As of SerialEM 3.9, 6/14/21, the arrays of tilt series parameters, montage parameters, and file options are saved to the Navigator file.  Thus it is possible to exit SerialEM, restart, and reload a Navigator file with all of these settings.  However, imaging states are not saved.

Stopping and intervening: When running tilt series from the Navigator, it is still possible to intervene manually, but the situation is somewhat complex because of the two layers of control involved. There are three distinct phases where the End and STOP buttons in the Camera and Script control panel have three different effects.

1. During the actions run by the Navigator prior to the start of the tilt series, such as rough eucentricity, the STOP button will stop the action and stop the Navigator. The current item will still be marked for tilt series acquisition, and you can resume from the Navigator Acquire dialog box . The End button (labeled End Nav) will signal the Navigator to end after this tilt series is finished.
2. During the startup phase of the tilt series, before the first image of the series is acquired, the STOP button will end the current operation and put the TSC into a 'postponed' state. When resumed from this state, the TSC will go through the whole sequence of startup operations again. Pushing the Resume button will open the Tilt Series Setup dialog box , from which you can restart the series. Alternatively, you can abandon this location and go on to the next tilt series by terminating the series, either from the Tilt Series menu or by pushing the End button (now labeled End TS). If you want to end all acquisitions, then select 'End Acquire' in the Navigator Menu BEFORE terminating the tilt series. During the startup phase, pressing the End button (labeled End Loop) will cause the TSC to stop after the saving the first image of the series.
3. Once the tilt series is through the startup phase, STOP stops the current operation and End (labeled End Loop) stops it at the end of a loop. At this point everything works as in a manually started tilt series. The Resume and Back Up buttons function as usual, opening their respective dialogs. The Tilt Series Setup dialog box can be opened to adjust parameters and resume from there. The tilt series can be terminated from the Tilt Series menu or with the End button (labeled End TS). Again, if you want to end all acquisitions, first select 'End Acquire' in the Navigator Menu.

Step-by-step Procedures: There a several ways to use the various tools provided for setting up multiple tilt series; here is one variation.

1. Make whatever maps are needed to locate your areas of interest. This could be just a low-mag map, or a low-mag map plus a series of medium-mag maps. The steps below for making anchor maps would not be needed if all points are located in medium-mag maps.
2. Open a file for maps of the areas to be acquired.
3. Locate the first area of interest. Run the Rough Eucentricity procedure or otherwise adjust the Z height to be approximately the eucentric height.
4. Go to the magnification required for the tilt series and acquire an image of exactly the area to be imaged.
5. Save this image and make it a map.
6. Go to a magnification where the camera field of view is 10-15 µm (if you have a state set up for this in the Imaging State dialog, set this state there).
7. Acquire an image and save it as a map.
8. Check 'For anchor state' in the Navigator dialog.
9. Return to the working magnification (use 'Restore Prior State' in the Imaging State dialog).
10. Run the Tilt Series Range Finding procedure or otherwise determine the limits of the series.
11. Select the first map again and check 'Tilt series' in the Navigator.
12. Define file properties as needed then define tilt series properties.
13. Go to the next area of interest, and adjust the eucentric height approximately if necessary.
14. Acquire an image of the area to be acquired.
15. Press the Anchor Map button. This will save the first image as a map and make a second map for an anchor.
16. Determine the angular range for the series.
17. Select the higher mag map again if necessary and check 'Tilt series'. This time no dialogs will open.
18. Press the TS Parameters button to import an angular range determined with the Range Finder and to adjust any other parameters
19. To adjust the automatically generated filename, press the Filename button.
20. Continue with steps 13 to 19 for any other areas to be acquired.
21. Check that beam autocentering is working correctly, if you plan to use it.
22. If appropriate, check the parameters for specimen cooking.
23. Select 'Acquire at Items' from the Navigator menu. Tilt series acquisition will be selected as the action to perform.
24. Select 'Autocenter beam' if you are set up to use it; 'Realign to Item' to align to the maps at each position; 'Cook specimen' if appropriate; and 'Fine Eucentricity if this operation is not selected in the tilt series parameters for each series
25. Set 'Close valves at end' and 'Send email' as desired, then press the GO button.

The order of the steps can be varied. If you adjust eucentric height after making a map item, you can press the Update Z button in the Navigator to change the Z value of the map item. If you run the Range Finding procedure after setting other tilt series parameters, press TS parameters to get the new angular limits into the tilt series parameters.

If you are acquiring montaged tilt series, you may want to make the map at the working magnification be a montage of the same size, although this is not essential. In this case, you would have two files for the maps, one for these montages and one for the anchor maps. A procedure for doing this would be as follows:

1. Open a montage file for the maps at the working magnification.
2. Locate the first area for acquisition.
3. Press Prescan in the Montage Control panel to take a trial montage rapidly.
4. Shift the prescan with the right mouse button and take another if necessary.
5. Take a full montage and make it a map.
6. Open another file for single frame images.
7. Go to a magnification appropriate for taking an anchor map.
8. Take an image and save it as a map. Mark this item 'For anchor maps'.
9. Return to the working magnification.
10. Select the montage file to be the current file, or just close the second, single-image file.
11. Perform other tasks for setting up a tilt series at this location, as above
12. Locate the next area for acquisition.
13. Use Prescan to localize the desired area, then take a full montage.
14. Press Anchor Map.
15. Perform other tasks for setting up a tilt series at this location.
16. Repeat steps 13 to 15 for further areas to be acquired.

Note that the Anchor Map button becomes particularly convenient when maps are kept in multiple files.