MicroED data collection with SerialEM

We developed a procedure for the cryoEM method MicroED using SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. More than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.

A maximum of ~140° of data can be collected per crystal (+/-70° tilt) so that, depending on symmetry, a single nanocrystal may yield an almost complete dataset 5 . Data from several crystals can be merged to increase completeness if each crystal is oriented differently on the grid 6 . MicroED data is then processed using standard X-ray crystallography software where structures are determined and refined as described before 7,8 .
Until now, all MicroED data were collected manually. Thus, the method could benefit from automation, where multiple targets are selected for acquisition in a single run, as is commonly done with other cryoEM modalities such as electron tomography (ET) 9 and single particle analysis (SPA) 10 . These methodologies utilize software that controls the electron microscope and camera in order to perform the operations necessary during long-running data acquisition sessions with minimum human intervention. Automation is attractive because it increases the throughput of the instrument, which is particularly relevant to busy facilities where microscopes are operated as shared resources. It also reduces error arising from manual execution of repetitive tasks and allows for data collection 24/7. It is compatible with modern electron microscopes and imaging detectors from several major manufacturers, and provides a consistent user interface across different hardware platforms. The software is highly extensible and allows microscope and camera acquisition tasks to be automated through its scripting command processor. Updates to the software and user-created scripts have enabled increasingly automated SPA 12 and ET 13 data collection. Other TEM automation software programs exist primarily for imaging mode only 10 (e.g., for SPA and ET); SerialEM is currently the only one of these with the ability to control the microscope in diffraction mode in conjunction with a continuously-rotating stage.
We have developed a procedure using a script for SerialEM which enables largescale MicroED data collection on TEMs by Thermo Fisher Scientific (formerly FEI Company, Philips Electron Optics) coupled to electron detectors from various manufacturers. An overview of this procedure is presented in Figure 1 and generally follows a similar protocol commonly used for collecting SPA data. Once a grid containing nanocrystals is loaded into the microscope, the operator identifies potential crystals either by inspecting the grid manually using low-dose imaging 14 , or by montaging a whole-grid atlas. An atlas provides a fast way to visually screen an entire grid for crystals offline and keeps the exposure of the sample at a minimum. The location of each crystal is then marked in a similar way to selecting regions of holes/gridsquares for SPA data collection. The operator can then collect a sample diffraction pattern from each of the marked crystals to narrow down the selection of crystals for complete data collection (Figure 2). This step can be done either manually or automatically in SerialEM using its Navigator tools. Before data collection can begin on a selected set of crystals, the eucentric height needs to be determined at each data collection point on the grid. Akin to crystal centering in X-ray diffraction, this ensures that the crystal does not move out of the confines of the measurement during the continuous-rotation experiment 2 . Several reviews and papers have been published on crystal identification, troubleshooting, and microscope setup for diffraction 2,7,[15][16][17] . The stage coordinates of the selected, well-diffracting crystals are then loaded to the automated data collection pipeline.
Our pipeline coordinates all activities (microscope, crystal, and camera) to automate data collection. The operator inputs the desired rotation angle span (a range from -70° to +70° at maximum), rotation speed (degrees per second), exposure time per image frame (seconds), and destination directory for the data output; these are applied to all crystals on a job list. Data collection then proceeds in batch, where the SerialEM script directs the microscope sequentially through the list of selected crystals, one crystal at a time. SerialEM synchronizes crystal rotation and the start of data collection, such that the crystal is rotating at the desired, constant rate when the first frame is recorded. The entire rotation range is recorded as set, then camera recording stops,

Figure 1 | SerialEM automation workflow for MicroED.
A cryoTEM grid containing protein nanocrystals is examined via a whole-grid atlas collected at low magnification. Crystal areas are then identified from this atlas and added into the SerialEM Navigator queue for subsequent diffraction screening. Diffracting crystals on this list are selected for subsequent MicroED data collection in batch. a, b, c, Crystals selected for diffraction screening.   diffraction beam setting as mentioned above for crystal exposure. The selected area (SA) aperture was inserted, the size of which was set to accommodate the dimensions of the largest crystal chosen for diffraction. We then used the "Acquire at Points" function to acquire diffraction pattern exposures at each previously-saved crystal coordinate. For those crystals which showed protein diffraction, the position was revisited to set the crystal at eucentric height using the microscope controls, then queued in the Navigator for batch MicroED data collection.
MicroED data collection. Microcrystal coordinates prepared for data collection were set to "Acquire" in the SerialEM Navigator in the previous section. Before starting, the parameters for data collection, particularly output directory, rotation speed, angular range, frame exposure time, and frame binning, were configured in the CRmov script for

Introduction
This protocol describes the setup for continuous-rotation MicroED 1-10 crystal screening and automated multiple-crystal batch data collection using SerialEM 11 . The script "CRmov" described here will work with the Tecnai, Titan/Krios, and Talos series microscopes available from Thermo Fisher Scientific. Note that we use the phosphor screen for diffraction setup and screening, not the primary imaging detector; however, it may be desirable to use the detector for samples with very weak diffraction. If you do use the imaging device for these cases, do so with care and caution, as a focused high-energy direct beam may harm the detector. Also, we do not mention when to open or close the column valves, or whether to use a beamstop in this procedure; it is up to the user to use best practices when controlling dose to the sample and imaging detector. Our practice is to keep column valves closed or pre-specimen shutter inserted (beam blanker on) until absolutely necessary to avoid accumulating electron dose on the grid sample. We generally use a beamstop to prevent the direct beam from striking the detector surface.
This procedure assumes that the TEM has been optimally aligned for imaging in the SA lens magnification range, and SerialEM has been installed with proper microscope and camera communications as well as necessary stage and beam shift calibrations. It also assumes that the operator has a basic understanding of SerialEM, including the use of the Low Dose Control, the Navigator, and Scripts. We recommend the user to be familiar with the protocols for MicroED 3,6 prior to setting up SerialEM.

Microscopy
• intensity or illuminated area).
3. Insert beamstop and center the target area with the detector as necessary/possible.
Warning: Please be advised that the next step involves focusing the beam to a coherent spot; you must take appropriate precautions to protect your imaging detector from excessive radiation before continuing. 4. Please see warning in step A3 above before proceeding. Use the focus knob in diffraction mode (diffraction focus) to condense the beam to a coherent spot (smallest diameter), then move it behind the beamstop using diffraction shift. This beam is now set up in the proper diffraction condition for data collection. To save the beam setting in SerialEM, uncheck the "Continuous update" box.
5. Tip 1: Make sure that the objective aperture is in the "out" position.
6. Tip 2: You may need to adjust the diffraction stigmator as necessary to ensure the beam is round; this should be done using the microscope controls.

Tip 3:
If the beam is sweeping as you move through crossover, you must center the condenser aperture(s).  m. Add the current crystal position to the Navigator by pressing "Add Stage Pos" in the Navigator window ( Figure 2).
n. Set the Navigator point to "Acquire".
o. Repeat the steps B1c-n above for the next crystal.
p. After your crystal positions are set, and SerialEM is properly set to run CRmov (steps C1-3 below), you may edit the CRmov script to adjust batch collection parameters (step C4 below), then set up and run the batch data collection as specified in step C6 below.
2. Map-assisted search. This allows offline searching for crystals using a lowmagnification atlas of the entire grid. This procedure assumes that the beam image center (image shift) at low magnification is aligned to, or close to, the optical axis in the microscope. Otherwise, you will not be able to target the proper gridsquare/crystal in diffraction mode; or you must apply an offset to the crystal in View mode for proper targeting. b. Using the "Set up full montage" feature in the Navigator menu, collect a whole-grid atlas (also called "full montage" or "Low Mag Montage/LMM") of the grid by using the "Setup full montage" function in the Navigator menu.
Consult the SerialEM documentation for more information on setup.
c. Add crystal targets using the Navigator. Use the Navigator and whole-grid atlas to add points of crystal areas you wish to target/check for diffraction. f. Choose a saved crystal coordinate in the Navigator and press "Go To XY".
The microscope will move the stage to the selected target. l. After your crystal positions are set, and SerialEM is properly set to run CRmov (steps C1-3 below), you may edit the CRmov script to adjust batch collection parameters (step C4 below), then set up and run the batch data collection as specified in step C6.
m. Check each Navigator point for diffraction: batch mode. Continuing from step B2d above, make sure that View and Record modes are set for diffraction as described above in steps A1-4.
n. Choose a saved crystal coordinate in the Navigator and press "Go To XY".
The microscope will move the stage to the selected target.
o. Center a crystal in the SA aperture. Use detector-assisted live imaging (step B1a above) or the phosphor screen (step B1b) to make sure that the crystal is in view, then insert the SA aperture. Using the microscope stage controls, move the stage so that the crystal is centered in the beam, which is 12 now limited by the SA aperture. You may need to insert and remove the SA aperture as necessary to locate the crystal.
p. Make sure that the current crystal coordinate (from step B2n) is highlighted.
Delete this coordinate from the Navigator by pressing "Delete Item". Then, press "Add Stage Pos" to add this crystal's updated (x,y,z) coordinates to a new Navigator point. Repeat steps B2m-p for each crystal in the Navigator.
When done, continue to step B2q below.
q. In the Navigator, set the updated points you wish to test for diffraction (corresponding to the selected crystals) to "Acquire".
r. Adjust the camera parameters for Record mode to your desired exposure setting (e.g., 5 s) for diffraction mode.
s. Open a new file to save images by going to "File > Open New…".
t. In the SerialEM menu, go to "Navigator > Acquire at Points…". In the "Acquire at Items" window that appears (Figure 3  C. Continuous-rotation MicroED data collection using CRmov. A SerialEM script called "Continuous Rotation movie acquisition", or CRmov, was developed to automatically tilt to the starting angle, start continuous stage rotation, and continuously collect exposures until the end angle, saving these to files within a specified directory on disk. The resulting movie can then be converted to a standard crystallography format using tools 3 available at https://cryoem.ucla.edu/pages/MicroED for subsequent processing. This section will describe settings that should be checked and changed as necessary for each batch data collection session. These settings are in the "CHANGEABLE SETTINGS" section at the top of the script as seen in Figure 4. It is important to note that all points set to Acquire during a single run will use the settings entered in the script. If you wish to use different values for certain data collection parameters, such as path to root directory, angular range,  6. Using CRmov: Start a batch data collection run. This process assumes that SerialEM is properly configured to run CRmov (steps C1-3 above) and CRmov script parameters are set as necessary for the crystal coordinates in this batch run (steps C4,5 above). The script will override settings in SerialEM Camera Parameters to collect data at the binning and exposure time set in the script as described above in step C4. The steps below follow from either step B1p, B2l, or B2v above, where you should already have crystal coordinates, at eucentric height and ready for continuous-rotation MicroED data collection, set to "Acquire" in the Navigator.
a. Make sure that the beamstop is inserted (if necessary) before starting.
b. Ensure that the fullPath parameter is set properly in the script. This entry should be a directory on the local computer (i.e., local to SerialEM); refer to step C4a above for more information.
c. It is recommended to open the Log window (from the SerialEM taskbar menu: File > Open Log) to monitor progress and save as a reference for processing.
d. In the SerialEM taskbar menu at the top of the screen, choose "Navigator > Acquire at Points…". A dialog box titled "Acquire at Items" opens.
e. In the "Acquire at Items" dialog window, in the "Primary Task" section, select "Run script" then in the drop-down menu beside it, choose the CRmov script.
Refer to Figure 3 for reference. Click "GO" to start.
f. For each crystal position (i.e., each point in the Navigator set to Acquire (A), the batch run will create a subdirectory within the specified fullPath (refer to step C4a above). The process creates sequential TIF files of continuousrotation MicroED data and a text file with the filename corresponding to the Navigator Point/Label number and file extension .idoc. This file is the IDOC file containing metadata information for each frame collected.
g. After data collection is complete, save the data in the Log window (File > Save Log…), and optionally save the Navigator file if needed.

Timing
Experiment timing depends on crystal screening and data collection parameters, therefore it may vary greatly. In our tests using a Thermo Fisher CetaD camera coupled to a Thermo Fisher Talos Arctica TEM, a single crystal dataset, collected using a rotation sweep of +30° to -30° at a rotation speed of 0.5° s -1 , consisted of 200 frames of 4K × 4K data at 2 s per exposure and took 4 minutes to complete.

Code availability
The CRmov script is available as a Supplementary Document.

Supplementary Document
CRmov.txt is a script for SerialEM which can be imported into SerialEM's Script Editing Window. Updated versions of this script will be deposited to the SerialEM Script Repository