Step 1: Initial Setup

• Write down chamber pressure.
• Fill liquid nitrogen to the tank.
• Cool down the heat exchanger.
• Set N₂ gas flow rate to 180 mg/s.

Step 2: Chamber Check

• Check if there is any sample in the chamber.

Step 3: Grid Loading

• Wait 1 hour after cooling down, then load grids.

Step 4: System Wake-up and Beam Alignment

• Wake up the system.
• Turn Beam On.
• Perform 180° scan rotation for both holders.
• After rotation, ensure the slot is positioned at the bottom of the grid.

Step 5: Grid 1 Mapping Preparation

• Navigate to lowest magnification.
• Press F6 to enable live view.
• Switch to higher magnification.
• Perform coarse focus.
• At 2000x, do fine focus.
• Perform Sigma X and Y alignment.

Step 6: Z-Height Calibration

• In live mode, select Link Z to Forward.

Step 7: Project Setup in MAPS

• Open MAPS.
• Create a new project.
• Change save location to:
  \\AS-SPC\SharedData\Chen\20241106

Step 8: Grid 1 Pre-GIS Mapping

• Go to lowest magnification.
• Take a snapshot.
• Add tiles.
• Rename to “Grid1-preGIS”.
• Click Run.

⚠️ Repeat Steps 5–8 for Grid 2.

Step 9: Cryo-CLEM Data Integration

• Import cryo-CLEM data.
• Align the data.
• Add lamella positions.

Step 10: Sputter Coating Preparation

• Increase N₂ gas flow to 210 mg/s.
• Perform Sputter coating using Setting 1 for 15 seconds.

Step 11: GIS Deposition on Grid 1

• Apply GIS to Grid 1 for 10 seconds.

  ⚠️ Do not exceed 10 seconds. A thick GIS layer will hinder milling efficiency.

Step 12: Post-GIS Coating

• Perform Sputter coating again.
• Reset N₂ gas flow to 180 mg/s.

Step 13: High-Resolution Imaging (Grid 1)

• In XT-UI, take a high-resolution image.
• Save as: Grid1_afterGIS-XXX.tif.

Step 14: AutoTEM Setup

• Open AutoTEM.
• Change project name.
• Do not change save location (keep as D:\AuToTEM Cryo\Projects).

Step 15: (intentionally left blank for future use)

Step 16: Lamella Preparation

• Start lamella preparation.
• Manually adjust lamella placement.
• Proceed with milling and thinning step-wise.

Step 17: Manual Fine Polishing

1. In AutoTEM, select the lamella → Right click → Go to lamella position.
2. Increase tilt angle by +0.5° (e.g., T = 16.5 if stage = 16°).
3. Create a rectangle milling pattern, set direction: top to bottom.
4. Adjust brightness and contrast using a pattern at the side.
5. Position milling pattern on top of the lamella.
6. Start milling with 30 pA ion beam.
7. Use image shift Y to gently move the lamella into the milling pattern.
8. Check lamella thickness with SEM view (2 kV).

   A thin lamella should appear black in SEM.

Step 18: Documentation

• In MAPS:
  File → Save screenshot as → Save grid image with lamella positions.
• In XT-UI:
  • Take SEM and FIB images after focus adjustment.
  • Use the finest photo button.
  • Save images as:
    • L2_i.tif
    • L2_s_2kV.tif
    • L2_1200x_2kV.tif (for LM correlation)
• Go to lowest magnification → Take full grid image with SEM.
• Copy entire data folder to the lab server.

Step 19: Shutdown Procedure

• Put the system to Sleep.

  ⚠️ This is important to prevent ion beam waste.

• Reset N₂ gas flow rate to 12 mg/s.