Cassette position 1 is dedicated to the Cross Grating Grid

Grids are loaded with the C-clip toward position 1

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).

Menu: Navigator > Open

If the Imaging states window does not open with the Navigator window go to Navigator > Open imaging states

Menu: Settings > Open > Platform_Settings_SPA

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.

Double click on each grid map in the navigator window

Decide the grid you wish to screen first

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

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.

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.

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.

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.

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.

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.

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.

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.

Add Points on the desired holes

Stop adding and Collapse

Press A key to define acquisition at all points and Collapse again

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