The setup of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. This method enables fast setup of large-scale automated tomography data acquisition to maximize the time efficiency of tomography experiments that require a careful selection of acquisition targets. Begin by opening a dummy SerialEM instance.
Once the first grid square is mapped, use the SerialEM navigator menu option merge to see the montage in the dummy SerialEM instance. Double-click on the navigator window to open the grid square map. Search the map and use the dummy SerialEM navigator option add points to add image acquisition points on the target of interest.
After mapping the new squares, save the navigator file and merge the navigator again. Continue until all grid squares are mapped. Again, merge the navigator file with the dummy SerialEM instance.
Run the Pi EM virtual maps script from the dummy SerialEM menu. Select tools and select virtual anchor maps. This may take some time depending on the size and amount of the grid square maps, as well as the binning of the view and preview maps.
To ensure proper microscope performance, use the same magnification and beam size setup for data acquisition in the following order. Run SerialEM coma-free alignment by CTF. Insert and center an objective aperture.
Run SerialEM correct astigmatism by CTF and GIF quick tune. In SerialEM, open the new navigator file. De-select all A points.
Select the first view maps. Select collapse. Click on A twice and de-select collapse.
Select the first view map position. Press Shift T, then select the very last view map position and press Shift T again. Choose single frame images in the properties of the file to open a dialog.
In the next file properties dialog, select the desired parameters according to the imaging needs and the instrument setup. When prompted, give a name with a number and click on save. Set up the tilt series controller for the first TS position.
When done, click OK to set these parameters for all tilt series after this acquisition item. All preview maps are now selected as TS with a numbered filename. If needed, set the focus/track distance for each target.
Double-click on the view map to load it. Select the preview map in the navigator list. Then select edit focus in the navigator window.
In the low-dose control panel, de-select rotate inter-area axis to position trial and focus along the stage tilt axis. Click on the desired region in the loaded view map to set the focus trial position for this tilt series and ensure that the navigator item has TSP set. Repeat the procedure for all items.
Edit the focus range in the script PreTomo which runs before each tilt series. In the SerialEM tilt series menu, check run script in TS and select the script number of the DuringTomo script during each tilt. Check the nitrogen tank level and whether the auto-loader turbo off is selected.
Check the data storage free space. In the SerialEM file menu, de-select continuous saving for the log file. Each tilt series will get its own log file.
After clicking on acquire at items in the navigation menu, run the script PreTomo. Select primary task acquire tilt series, then select run script after PostTomo. Select close column valves at end and send email at end, then click on go.
71 suitable squares were selected on the grid map at lower magnification. Medium magnification maps were acquired with settings that allow for the direct visualization and identification of the sample of interest, coronaviruses in this case. The acquisition time was three minutes per square, three hours and 45 minutes in total.
As the first square map was created, a dummy SerialEM instance was opened on a separate computer to visualize the square map and to add points on targets suitable for tilt series acquisition. SerialEM low dose was set up and reference view and preview images were taken and saved as maps. The latter maps could then be used immediately on the dummy SerialEM instance to generate the virtual view and virtual preview from the corresponding square map images.
The virtual view maps were used for an initial centering of the target, followed by a final centering performed at the actual tilt series acquisition magnification using the virtual preview map. Users should allow themselves plenty of time to explore the sample landscape. After having searched some maps, one develops a better feel for good quality areas to collect data from.
