1. Pre-Experiment Preparation

a. Transfer Shuttle Drying

• Dry the Transfer Shuttle on the Leica Cryo Tool Dryer (CTD) for at least 20 minutes before use to reduce contamination.

b. Cryo Stage Heating

• Run stage heating for 30 minutes at 60°C:
  • Do not connect the LN₂ hose.
  • Lift the objective to the uppermost position (fully retracted).

2. Power On Sequence

a. Microscope

• Turn on the microscope, the control panel and the pump.
• Turn on the camera.

b. Laser

• Important: Turn on the front switch of the laser before the back switch.
  • Otherwise, the laser will not power on.

3. Launch Imaging Software

• Start LAS X software.

4. Initialize Motorized Stage

• Click Initialize Stage.
• ⚠️ Do not block the stage — allow it to move freely during initialization.

5. Select Imaging Mode

• Navigate to:
  MatrixScreener → Matrix MAPS + CLEM

6. Fill Liquid Nitrogen (LN₂)

• Fill LN₂ into the storage tank.
• ⚠️ Do not fill all the way to the top:
  • LN₂ may spill when inserting the heat exchanger.

7. Connect Cryo Hose to Pump and Stage

a. Start the Pump

• Press COOL to start.

b. Wait for Purge

• Wait until the hose is purging nitrogen gas.

c. Connect Hose to Cryo Stage

• Insert the hose into the cryo stage.
• Lock the hose in place:
  • Push the locking screw toward the hose while tightening.
  • ⚠️ If not pushed first, the hose may be pulled out easily and won’t seal properly.
    • Moisture may enter and condense, blocking the view.

8. Wait for Stage Cool Down

• When temperature reaches -195°C, wait another 15 minutes before loading the sample.

9. Cool Down the Transfer Shuttle

a. Setup

• Ensure the transfer shuttle shutter is closed.

b. Insert Cartridge

• Place an empty cartridge into the loading position.
• Close the slider.
• Turn the rod to the open-O position.
• Cover the cartridge with the shielding.

c. Fill LN₂

• Cover the transfer shuttle with its lid.
• Open the rotatable opening.
• Fill LN₂ inside the shuttle.
  • ⚠️ Do not overfill — avoid pouring over the rod.
  • Otherwise, the rod may freeze and become immobile.
• The shuttle is fully cooled when the LN₂ stops boiling inside.

10. Load AutoGrids into Cartridge

• Proceed with loading autogrids into the pre-cooled cartridge.

11. Load Grid into Microscope

a. Ensure Lens Is Up

• ⚠️ Objective lens must be lifted to avoid damage from the shuttle.

b. Stage Access

• Open the stage slider.

c. Insert Transfer Rod

• Insert the transfer rod fully.
• Rotate to Open position to release the cartridge.
  • ⚠️ Must insert all the way to the end.
  • Otherwise, you won’t be able to focus on the grid.

d. Retract Rod

• Pull the rod back.
  • The cartridge should remain in the stage.

e. Clean Up Shuttle

• Remove the transfer shuttle from the stage.
• Pour out any remaining LN₂.
• Place it on the Cryo Tool Dryer (CTD) for baking out.

12. Imaging Procedure

a. Load Experiment

• Open LAS X and load your experiment.

b. Move to Left Grid

• In the MatrixScreener window:
  • Right-click the left grid.
  • Select “Move stage to left grid center”.

c. Autofocus

• Run Auto Focus.
• ⚠️ If autofocus fails, adjust manually until sharp.

d. Spiral Scan

• Run the Spiral Scan to locate good regions for imaging.

e. Focus Map Setup

• Select a circular imaging area.
• Click on several points within the circle to define a focus map.
• Click Start Focus Map to collect Z positions.

f. Mosaic Scan

• Enable 4 channels.
• Adjust the Z-stack range according to your sample thickness.
• Click Run Matrix to begin automated imaging.

g. Export Data

• After acquisition is finished, export data files for downstream analysis.

13. Finishing Up

a. Close Software

• Exit the LAS X software and any other imaging applications.

b. Retrieve Grids

• Lift the objective lens to its uppermost position.
• Retrieve the cryo grids from the stage.

c. Heat the Stage

• On the temperature control box, press “Heat”.
• Wait until the stage heats to 60°C.

⚠️ Do not remove the hose before the temperature reaches 60°C.
Removing the hose prematurely can cause damage due to freezing.

d. Disconnect the Cryo System

• Once the stage is at 60°C:
  • Remove the LN₂ hose.
  • Remove the pump from the LN₂ tank.

e. Return Grid Boxes

• Place the used grid boxes back into the LN₂ storage tank for long-term storage.