1. Introduce your grids in the cassette

   • Cassette position 1 is dedicated to the Cross Grating Grid
   • Grids are loaded with the C-clip toward position 1

2. Load the cassette in the NanoCab. Make sure no contaminant (fiber, hair) entered the NanoCab.
3. Check Autoloder temperatures, then load the NanoCab in the microscope

   

1. a.Make sure HT and FEG are on

* Under « setup » tab, « high tension » and « operate » are highlighted yellow b. Make sure that column valves are closed and turbo pump is on i. Under « setup » tab, « col. Valves closed » and « Turbo on » are highlighted yellow c. Make sure that EM column vaccum is stable (IGP1 -6) d. Make sure that Stage neutral i. Under « Search » tab 1. click « X,Y » button to zero out the X, Y to center the stage 2. If necessary Click « AB » button to zero out  coordinates of the stage e. Make sure cold trap already with LN2, refill every 3-4 hours

2. Load grid in holder a. Place holder in stand with spring holder up b. Lift (open) spring holder using metal pin c. Center specimen grid in holder socket d. d. Close spring holder using metal pin

1. Start Serial-EM software

   • On the microscope computer, turn on the SerialEM server (if not already running) .
   • On the camera computer, turn on the serialEM install that match your method (SPA or uED).

2. Run the master script

   • In serial-EM: script tab > run > Master (starts automatically with serial-EM).
     • Select your directory in /data/users/your_directory/ and create your working directory
     • Position of the first grid = position in the cassette where you loaded your first grid (This should be at least position 2, since position 1 is occupied by the Cross Grating Grid)
     • Do you want to screen ? Yes
     • Number of Grids = the number of grids you have loaded in the cassette
     • Provide all grid names one by one
   • Menu: Navigator > Open
   • If the Imaging states window does not open with the Navigator window go to Navigator > Open imaging states

3. Load platform or user settings

   • Menu: Settings > Open > SerialEM_Settings_SPA

1. Setup the microscope for recording the Atlases

   • On TEM user interface (TUI).
   • Autoloader or Tune tab > Apertures panel : select condenser 2 150μm. Make sure Objective and selected area apertures are out (None status).
   • Camera tab > CCTV/Camera panel > Shutter: make sure “Standalone Camera” is yellow. Otherwise, press on it.
   • On Serial-EM.
   • Imaging States window > double click on Atlas C2 150 μm.
   • On the microscope computer, make sure beam settings were updated according with Atlas imaging state.

2. Setup montage parameters

   • in Navigator tab > Montaging & Grids > Setup Full Montage
     • Make sure magnification is set to 34X.
     • Make sure binning is set to 2.
     • On Glacios + K2,number of pieces should be 6*6 with an overlap of 15%-20%.
     • Make sure other settings are in agreement with the Montage setup window bellow.
     • Press OK > The file Properties window will open.
     • If you are screening more than 10 grids, change 360 to 3600 in Maximum number os sections.
     • Make sure settings are in agreement with the image of the window bellow.
     • Press OK, then save the mrc file in the work directory with a meaningful name i.e. “Atlases.mrc”.

3. Manually collect atlases or run the gridmaps script

   1. Optional: Manually run cassette inventory

      • After grid introduction, the autoloader initialization takes a few minutes.
      • in the TUI, Make sure Autoloader elements temperatures are colder than -170°C. This is particularly important for the cartrige gripper, the autoloader element which grap the specimens.
      • in the TUI, press on Inventory button and wait until autoloader finishes the inventory. If you did not load a full cassette, you can press on stop inventory once the last loaded position was mapped.

   2. Automatically collect atlases with the gridmaps script

      • To fasten Atlases acquisition, turn on turbo always on in TUI.
      • In Serial-EM Script tab > run > gridmaps.
      • The interface will ask three questions:
        1. Did you insert 150 μm C2 aperture ? : if not, refers to section 3.1, then press Yes.
        2. Please switch on the 'Standalone camera' in the camera/shutter menu in - only press 'OK' if it's done: In TUI, buttom must be yellow. Otherwise, press on it.
        3. Do you want to launch the inventory with a 16min delay ? : Look at the autoloader temperatures as indicated in section 3.1.a. If Cartridge gripper is colder than -170°C, press No. Otherwise, wait for temperature to be cold enough OR press Yes.
      • Wait to see the few first Atlas tiles.
      • Doing the Atlas for one grid takes approximately 10 minutes.

   3. Manual Atlas collection

      • This is to run a grid map on a single grid.
      • In the Montage Controls Panel, press Start.
      • Once the atlas run is done, make sure it had been saved as a map in the navigator windows. Otherwise, press New Map in the navigator windows.

1. Look at your Atlases and select the first Grid to screen

   • Double click on each grid map in the navigator window
   • Decide the grid you wish to screen first

2. Introduce the Grid in the column

   • In TUI, select the cassette position of the grid to be loaded, then press on Load.

3. In TUI, select "Turbo Auto Off (default)"

   

1. Setup the microscope for Squares imaging

   • On TEM user interface (TUI) : Autoloader or Tune tab > Apertures panel : select condenser 2 30μm or 50μm.
   • On Serial-EM : Imaging States window > double click on Square imaging state.
   • On the microscope computer, make sure beam settings were updated according to Atlas imaging state.

2. Open the column valves.
3. (Optional) Determine the overall eucentric height.

   • In Serial-EM, center a square in the middle of the grid.
     1. add a marker (green cross) by left-clicking on a square.
     2. in the navigator windows press the go to marker button. Wait for the stage to reach the specified position.
     3. take a record image.
     4. if the square is off-center: keep pressed the right mouse button, grab the center of the square to the center of the screen, and release the button. Wait for the stage to reach its position.
   • In Tasks tab > Eucentricity > press on Rough Eucentricity.
   • Once eucentric height was determined, in navigator windows, select the line of the corresponding atlas and press on Update Z button.

4. Correct for the coordinate discrepancy between Atlas and Square magnifications

   • In the Navigator windows, double click on the Atlas line to load it.
   • On the Atlas, look for the feature which is easily recognizable. It is recommended to choose a feature in the vicinity of the area of interest.
   • Add a Point on the feature

     1. In the navigator windows, click on Add Points. The button Will change to Stop Adding.
     2. Click on the chosen feature.
     3. In the navigator windows, click on Stop Adding.

   • Make sure the Point is selected in the Navigator windows.
   • In the Navigator windows, press on Go To XY, then wait for the stage to reach the feature position.

     

   • In the Camera & Script panel, press on Record.
   • Find the feature in the newly recorded image. If necessary, recenter the feature with the same method presented in section 5.2.d.
   • Add a Marker on the chosen feature, i.e. simply do a left click which will draw a green cross.
   • Make sure the Point is still selected in the Navigator windows.
   • In Navigator tab, click on Shift To Marker…. A new windows will open, indicating the X and Y translation (in um) to apply to align the feature in at square magnification to the one at Atlas magnification. If this value make sens, i.e X and Y shifts are in the 10 → 50 um range, press OK.
   • If you did a mistake, like applying the shift with an atlas/square map selected instead of the point, the sift can be deleted in Navigator tab > Undo last Shift.

5. Select squares to be mapped.

   • Load the Atlas.
   • Add Points in the middle of all the squares that you wish to map.
   • Change Points status to Acquire.

     • In Navigator windows:
       1. Check the Collapse radio button.
       2. Select the group of items.
       3. Check the Acquire (A) radio button to activate the Points.
       4. Uncheck the Collapse radio button. All Points should display a A.

   • (Optional) Go through all the points to correct for centering.

     • In the navigator windows:
       1. Select the first Square Point.
       2. Press Go to XY button, then wait for the stage to reach the square.
     • In the Camera & Script Panel, take a Record.
     • If the Point if off-centered:
       1. Make sure the Point which is selected is the one you want to move.
       2. click on Move Item button in the Navigator windows. The button will change to Stop Moving.
       3. Click in the middle of the Square.
       4. Click on the Stop Moving button.
     • Repeat these steps for all the Square Points.

6. Automatically collect squares maps and determine their eucentric height.

   • Menu > File > Open New. Save a mrc file wherein Square maps will be saved.
   • In menu > Navigator tab > Acquire at Items: start square mapping

     1. Tick Acquire and save image or montage & Make Navigator map radio buttons.
     2. Make sure that IMAGES will be saved into the file: directs toward the previously saved mrc file.
     3. Tick the Rough Eucentricity option and make sure it will be performed at every item.
     4. If you plan to leave the microscope room while it records squares maps, tick the close column valves at end option.
     5. Press GO.

1. Setup the microscope for Low Dose imaging

   • On TEM user interface (TUI) : Autoloader or Tune tab > Apertures panel : select condenser 2 30μm and Objective 100μm.
   • On Serial-EM : Imaging States window > double click on C2 30 Parallel imaging state.
   • Make sure the Low Dose panel is activated.

     

2. Correct for the coordinate discrepancy between Square and Low Dose View magnifications

   • In the Navigator windows, double click on a Square line to load it.
   • On the square, look for the feature which is easily recognizable.
   • Add a Point on the feature

     1. In the navigator windows, click on Add Points. The button Will change to Stop Adding.
     2. Click on the chosen feature.
     3. In the navigator windows, click on Stop Adding.

   • Make sure the Point is selected in the Navigator windows.
   • In the Navigator windows, press on Go To XYZ, then wait for the stage to reach the feature position.

     

   • In the Camera & Script panel, press on View.
   • Find the feature in the newly recorded image. If necessary, recenter the feature with the same method presented in section 5.2.d.
   • If you have trouble finding the feature, use the fluo-screen and the joystick:

     1. lower the fluorescent screen with the console button indicated in the lower panel of the TEM User Interface.
     2. with the joystick, move the stage so that the desired feature is in the green square (K2 area) on the fluorescent screen.
     3. Lift up the fluorescent screen with the same console button.

   • Add a Marker on the chosen feature, i.e. simply do a left click which will draw a green cross.
   • Make sure the Point is still selected in the Navigator windows.
   • In Navigator tab, click on Shift To Marker…. A new windows will open, indicating the X and Y translation (in um) to apply to align the feature in at square magnification to the one at Atlas magnification. If this value make sens, i.e X and Y shifts are likely lower than 10um, press OK.
   • If you did a mistake, like applying the shift with an atlas/square map selected instead of the point, the sift can be deleted in Navigator tab > Undo last Shift.

3. Correct for coordinate discrepancy between View and Record Magnifications.
4. Select the focusing area

   1. Center a hole. If you plan to record images at the hole periphery, center the camera closer to a hole edge.
   2. Take a View and correct position if necessary.
   3. In the Low Dose control panel:
      • Tick the Focus radio button (1).
      • Make sure Keep Focus and Trial Identical is not thicked (2).
      • Make sure Rotate inter-area axis is ticked (3).
   4. On the view image, left-click between four holes. This will move the focusing area to this place.
   5. Tick the None radio button to leave the focus display.

5. Create a reference hole

   1. Center a hole. It must be non-empty, with an ice thickness representative of your grid, and free of ice contamination. If you plan to record images at the hole periphery, center the camera closer to a hole edge.
   2. Take a View and correct position if necessary.
   3. Adjust the camera field of view to capture a single hole in the image.

      • Note: For gold grids, have a little piece of the surrounding holes help in downstream alignment.

      1. Press Setup in the Camera & Script panel.
      2. Tick the View radio button.
      3. Adjust the Camera field of view with the Area Size buttons.
      4. Press Acquire to take a snapshot and Adjust the area size if necessary.

   4. In the Buffers Control panel, press on P to save the reference hole image in this buffer.

6. Center the Objective aperture.

   • Note: Be very careful ! for this step, you will need to work in diffraction mode. Direct exposure of the camera sensor with the direct beam may result in damages of the electron detector.

   1. Center on carbon or gold support film.
   2. In the Low Dose Control panel, press the Rec. button to switch the beam from View to Record setup.
   3. Lower the fluorescent screen as described in section 6.2.
   4. On the microscope Console, press the Diffraction button.
   5. Recenter the direct beam in the small red circle of the fluorescent screen with multifunction X & Y knobs.
   6. With the mouse knob, change the sensitivity of the fluorescent screen to make the objective aperture shadow visible.
   7. In the TEM User Interface, on the Apertures panel, press the Adjust button next to the Objective aperture selector.
   8. With multifonction X & Y knobs, center the objective aperture around the direct beam.
   9. Deselect the adjust button in the Apertures panel.
   10. Turn off diffraction mode.
   11. Lift the fluorescent screen.

7. Center the low-dose beams on the camera.

   1. Move to a empty area (empty hole or brocken square)
   2. In the Low Dose Control panel, tick Continuous Update.
   3. Switch to record beam with the Rec. button.
   4. On the console, press the Eucentric Focus button.
   5. Lower the fluorescent screen.
   6. In TEM User Interface > Tune or Autoloader tab > Direct Alignments panel: press on beam shift.
   7. With multifunction X & Y knobs, center the beam on the fluorescent screen, then press Done.
   8. Switch to View beam with the Vie. button. Tick Set in Additional beam shift. View beam only need to be centered once.
   9. With the trackball, center the view beam on the fluorescent screen. Untick the Set button.
   10. Center the Record beam again as described above.
   11. Switch to Focus beam (Foc. button) and change its diameter with the intensity knob according with the desired focus surface (depends on your hole spacing).
   12. Center focus beam as explained for the view beam (with Set ticked and trackball).
   13. Switch to Trial beam (Tri. button) and change its diameter with the intensity. Trial beam will be used for auto-centering and its diameter should fit in the camera field (green square) on the fluorescent screen.
   14. Repeat Record (Beam Shift + M X/Y), Focus and Trial (Set ticked + Trackball) centering until all beams stay centered.
   15. Untick Continuous update on the Low Dose Control panel.

8. In an empty hole or a broken square: Menu > Scripts > run > DatasetDefineVacuumIntensity
9. measure the dose rate and set up record parameters.

1. Select a few target holes on each square

   • Add Points on the desired holes
   • Stop adding and Collapse
   • Press A key to define acquisition at all points and Collapse again

2. Manually screen your targets or run the script Screening Holes

   • Menu: Navigator > Acquire at Items > Screening-Holes (uncheck Rough Eucentric)

1. Double-click on the desired grid in the TUI
2. Go back to step 4

1. Save your Navigator
2. Close your Navigator
3. Load the Cross Grating Grid in the column (usually in position 1 of the cassette)
4. Remove the cassette with your grids from the autoloader in the NanoCab
5. Remove the cassette and your grids from the NanoCab