====== The Screening Pipeline ======




==== 1. Load  Grid ====

  - ++Introduce your grids in the cassette|<div>
  * Cassette position 1 is dedicated to the Cross Grating Grid
  * Grids are loaded with the C-clip toward position 1
</div>++ 
  - Load the cassette in the NanoCab. Make sure no contaminant (fiber, hair) entered the NanoCab.
  - ++Check Autoloder temperatures, then load the NanoCab in the microscope|<div>{{ :autoloader_tempertures_all.png?nolink |}}
</div>++






==== 2. Setup Serial-EM ====

  - ++Start Serial-EM software|<div>
  * On the microscope computer, turn on the SerialEM server (if not already running) {{ :fei_sem_server.png?direct|}}.
  * On the camera computer, turn on the serialEM install that match your method (SPA or uED){{ :serialem.png?nolink|}}.
</div>++
  - ++Run the master script|<div>
  * 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//
</div>++
  - ++Load platform or user settings|<div>
  * Menu: //Settings > Open > Platform_Settings_SPA//
</div>++ 


==== 3. Record Atlases ====

  - ++Setup the microscope for recording the Atlases|<div>
    * 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).{{ :apertures_1.png?nolink&400 |}}
    * 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//.{{ :platform_is.png?nolink |}}
    * On the microscope computer, make sure beam settings were updated according with Atlas imaging state.
</div>++
  - ++Setup montage parameters|<div>
  * 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.{{ :montage_window.png?nolink |}}
    * 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.{{ :montage_window_2.png?nolink |}}
    * Press OK, then save the mrc file in the work directory with a meaningful name i.e. "Atlases.mrc".
</div>++
  - ++Manually collect atlases or run the gridmaps script|<div>
  - ++Optional: Manually run cassette inventory|<div>
    * //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.{{ :autoloader_tempertures_cartridgegripper.png?nolink |}}
    * 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.{{ :autoloader_inventory_1.png?nolink |}}
</div>++
  - ++Automatically collect atlases with the gridmaps script|<div>
    * To fasten Atlases acquisition, turn on //turbo always on// in TUI.{{ :autoloader_turbo_on.png?nolink |}}
    * In Serial-EM //Script// **tab** > //run// > gridmaps.
    * The interface will ask three questions:
      - //Did you insert 150 μm C2 aperture ?// : **if not, refers to section 3.1**, then press Yes.
      - //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. {{ :standalone_camera_2.png?nolink |}}
      - //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.
</div>++
  - ++Manual Atlas collection|<div>
    * This is to run a grid map on a single grid.
    * In the Montage Controls Panel, press //Start//.{{ :montage_controls_panel.png?nolink |}}
    * 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.{{ :navigator_atlases_newmap.png?nolink |{{ :navigator_atlases.png?nolink |{{red|//Include here an image of the navigator, with map and button highlighted.//}}
</div>++
</div>++

==== 4. Select and Load a Grid ====

  - ++Look at your Atlases and select the first Grid to screen|<div>
    * **Double click** on each grid map in the navigator **window**{{ :navigator_display_gridmap.png?nolink |}}
    * Decide the grid you wish to screen first
</div>++
  - ++Introduce the Grid in the column|<div>
    * In TUI, select the cassette position of the grid to be loaded, then press on Load.{{ :autoloader_load_grid_1.png?nolink |}}
</div>++
  - ++In TUI, select "Turbo Auto Off (default)"|<div>{{ :autoloader_turbo_autooff.png?nolink |}}
</div>++

==== 5. Record square maps ====

  - ++Setup the microscope for Squares imaging|<div>
    * On TEM user interface (**TUI**) : Autoloader or Tune tab > Apertures panel : select condenser 2 30μm or 50μm.{{ :c2-30um_1.png?nolink |}}
    * On Serial-EM : Imaging States window > **double click** on //Square// imaging state.{{ :platform_is_square.png?nolink |}}
    * On the microscope computer, make sure beam settings were updated according to Atlas imaging state.
</div>++ 
  - Open the column valves.
  - ++(Optional) Determine the overall eucentric height.|<div>
    * In Serial-EM, center a square in the middle of the grid.
      - add a marker (green cross) by **left-clicking** on a square.
      - in the navigator **windows** press the //go to marker// button. Wait for the stage to reach the specified position.
      - take a //record// image.{{ :camera_and_script_panel_record.png?nolink&400 |}}
      - 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.
</div>++
  - ++Correct for the coordinate discrepancy between Atlas and Square magnifications|<div>
    * 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|<div>
      - In the navigator **windows**, click on //Add Points//. The button Will change to //Stop Adding//.{{ :navigator_add_points.png?nolink&400 |}}
      - Click on the chosen feature.
      - In the navigator **windows**, click on //Stop Adding//.
</div>++
    * 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.|<div>{{ :navigator_add_points_2.png?nolink&400 |}}
</div>++
    * In the //Camera & Script// **panel**, press on //Record//.{{ :camera_and_script_panel_record.png?nolink&400 |}}
    * 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//.
</div>++
  - ++Select squares to be mapped.|<div>
  * Load the Atlas.
  * Add Points in the middle of all the squares that you wish to map.
  * ++Change Points status to Acquire.|<div>
  * In Navigator **windows**:
    - Check the //Collapse// radio button.{{ :navigator_collapse.png?nolink&400 |}}
    - Select the group of items.
    - Check the //Acquire (A)// radio button to activate the Points.{{ :navigator_to_aquire.png?nolink&400 |}}
    - Uncheck the //Collapse// radio button. All Points should display a //A//.{{ :navigator_aquire.png?nolink&400 |}}
</div>++
  * ++(Optional) Go through all the points to correct for centering.|<div>
  * In the navigator windows:
    - Select the first Square Point.
    - 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:
    - Make sure the Point which is selected is the one you want to move.
    - click on //Move Item// button in the Navigator **windows**. The button will change to //Stop Moving//.{{ :navigator_move_item.png?nolink&400 |}}
    - Click in the middle of the Square.
    - Click on the //Stop Moving// button.
  * Repeat these steps for all the Square Points.
</div>++
</div>++
  - ++Automatically collect squares maps and determine their eucentric height.|<div>
    * Menu > File > //Open New//. Save a mrc file wherein Square maps will be saved.
    * ++In menu > Navigator tab > Acquire at Items: start square mapping|<div>
      - Tick //Acquire and save image or montage// & //Make Navigator map// radio buttons.
      - Make sure that //IMAGES will be saved into the file:// directs toward the previously saved mrc file.
      - Tick the Rough Eucentricity option and make sure it will be performed at every item.
      - If you plan to leave the microscope room while it records squares maps, tick the //close column valves at end// option.
      - Press GO.{{ :aquire_at_items_1.png?nolink |}}
</div>++
</div>++

==== 6. Prepare Serial-EM for screening ====

  - ++Setup the microscope for Low Dose imaging|<div>
    * On TEM user interface (**TUI**) : Autoloader or Tune tab > Apertures panel : select condenser 2 30μm and Objective 100μm.{{ :ld_apertures_settings.png?nolink |}}
    * On Serial-EM : Imaging States window > **double click** on //C2 30 Parallel// imaging state.{{ :platform_is_ld.png?nolink&600 |}}
    * ++Make sure the Low Dose panel is activated.|<div>{{ :ld_none_active.png?nolink |}}
</div>++ 
</div>++ 
  - ++Correct for the coordinate discrepancy between Square and Low Dose View magnifications|<div>
    * 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|<div>
      - In the navigator **windows**, click on //Add Points//. The button Will change to //Stop Adding//.{{ :navigator_add_points.png?nolink&400 |}}
      - Click on the chosen feature.
      - In the navigator **windows**, click on //Stop Adding//.
</div>++
    * 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.|<div>{{ :navigator_add_points_2.png?nolink&400 |}}
</div>++
    * In the //Camera & Script// **panel**, press on //View//.{{ :camera_and_script_panel_view.png?nolink |}}
    * 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:|<div>
    - lower the fluorescent screen with the console button indicated in the lower panel of the TEM User Interface.{{ :tui_lower_panel_screen_lift.png?nolink |}}
    - with the joystick, move the stage so that the desired feature is in the green square (K2 area) on the fluorescent screen.
    - Lift up the fluorescent screen with the same console button.
</div>++
    * 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//.
</div>++
  - Correct for coordinate discrepancy between View and Record Magnifications.
  - ++Select the focusing area|<div>
  - Center a hole. If you plan to record images at the hole periphery, center the camera closer to a hole edge.
  - Take a View and correct position if necessary.
  - 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).{{ :ld_focus_1.png?nolink |}}
  - On the //view// image, **left-click** between four holes. This will move the focusing area to this place.{{ :focus_are_annotated.png?nolink&400 |}}
  - Tick the //None// **radio button** to leave the focus display.
</div>++
  - ++Create a reference hole|<div>
  - 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.
  - Take a View and correct position if necessary.
  - ++Adjust the camera field of view to capture a single hole in the image.|<div>
  * **Note:** For gold grids, have a little piece of the surrounding holes help in downstream alignment.
  - Press Setup in the //Camera & Script// **panel**.
  - Tick the //View// radio button.
  - Adjust the Camera field of view with the //Area Size// buttons.
  - Press Acquire to take a snapshot and Adjust the area size if necessary.
</div>++
  - In the //Buffers Control// **panel**, press on **P** to save the reference hole image in this buffer.
</div>++
  - ++Center the Objective aperture.|<div>
  * **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.
  - Center on carbon or gold support film.
  - In the //Low Dose Control// **panel**, press the //Rec.// button to switch the beam from View to Record setup.
  - **Lower the fluorescent screen** as described in **section 6.2**.
  - On the microscope Console, press the //Diffraction// button.
  - Recenter the direct beam in the small red circle of the fluorescent screen with **multifunction X & Y knobs**.
  - With the mouse knob, change the sensitivity of the fluorescent screen to make the objective aperture shadow visible.
  - In the TEM User Interface, on the //Apertures// panel, press the //Adjust// button next to the Objective aperture selector.
  - With **multifonction X & Y knobs**, center the objective aperture around the direct beam.
  - Deselect the  //adjust// button in the //Apertures// panel.
  - **Turn off diffraction mode.**
  - Lift the fluorescent screen.
</div>++
  - ++Center the low-dose beams on the camera.|<div>
  - Move to a empty area (empty hole or brocken square)
  - In the //Low Dose Control// panel, tick //Continuous Update//.
  - Switch to **record beam** with the //Rec.// button.
  - On the console, press the **Eucentric Focus** button.
  - Lower the fluorescent screen.
  - In TEM User Interface > //Tune or Autoloader// tab > //Direct Alignments// panel: press on **beam shift**.
  - With **multifunction X & Y knobs**, center the beam on the fluorescent screen, then press //Done//.
  - Switch to **View beam** with the //Vie.// button. Tick //Set// in //Additional beam shift//. **View beam only need to be centered once**.
  - With the **trackball**, center the view beam on the fluorescent screen. **Untick** the //Set// button.
  - Center the Record beam again as described above.
  - 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).
  - Center focus beam as explained for the view beam (with //Set// ticked and trackball).
  - 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.
  - Repeat Record (Beam Shift + M X/Y), Focus and Trial (//Set// ticked + Trackball) centering until all beams stay centered.
  - Untick //Continuous update// on the //Low Dose Control// panel.
</div>++
  - In an empty hole or a broken square: Menu > Scripts > run > DatasetDefineVacuumIntensity
  - measure the dose rate and set up record parameters.

==== 7. Start screening and analyze the results ====

  - ++Select a few target holes on each square|<div>
  * **Add Points** on the desired holes
  * **Stop adding** and **Collapse** 
  * Press **A** key to define acquisition at all points and **Collapse** again
</div>++
  - Manually screen your targets or run the script ++Screening Holes|<div>
  * Menu: //Navigator > Acquire at Items > Screening-Holes// (uncheck Rough Eucentric)
</div>++

==== 8. Load the next grid ====

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

==== 9. End your screening session ====


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