TFS Autoloaders Operating Procedure

1. Purpose

1.1 To transfer clipped autogrids into a TFS autoloader cassette and use a NanoCab to transfer them into an autoloader system

1.2 To care for and maintain autoloader tools (autoloader cassettes, transfer stations, and NanoCabs)

2. Definitions:

2.1 An Autoloader system is a robotic system for loading cryo-EM grids into the microscope, in which the user inserts autogrids into a cassette that is loaded into the microscope using a retractable arm.

2.2 An Autogrid is a Cryo-EM grid secured into a C-clip using clip rings (and prepared by the user through “grid clipping”).

2.3 A NanoCab is specially made vacuum insulated capsule that holds a cassette and docks with an Autoloader.

2.4 Liquid Nitrogen (LN2) is a cryogenic liquid stored under pressure.

2.5 Definition of terms for tools/equipment can be found in Figure 1.

3. Supplies & Equipment 

  • PPE (BSL-1)
    • Laboratory Coat
    • Nitrile Gloves
    • Goggles / Safety Glasses
    • Cryogenic Gloves
    • Face Mask
  • Chemicals/Reagents
    • Liquid Nitrogen
  • Table-top Transfer Station assembled with Covers, Metal Block, and Cassette Gripper Handle
  • NanoCab and lid
  • Autogrid Cassette
  • Autogrid Tweezer
  • Cassette Tweezer
  • Standard Fine-tip tweezer
  • Autogrid storage boxes and lids
    • Containing vitrified samples on clipped grids
  • Autogrid box opening tool
  • Large Forceps
  • Hair dryer or heat block (if needed to dry tools)

4. Procedure

4.1 Loading the C-clips into the clipping tool

  1. Initial fill should be performed from the top, through the opening in the clear cover.
  2. Remove and replace white cover with each pour.
  3. Do not fill higher than the top of the metal block (fig 3).

4.2 Cool the NanoCab

  1. Replace the lid with each pour to mitigate frost buildup.
  2. Be mindful not to overfill above the bottom of silver ring (fig 4).

The pin’s operation is essential for docking and undocking cassettes from the microscope. If the pin is stuck, notify center staff to get a different NanoCab until the defective one is repaired.

4.3 If Cassette is in the Microscope: Retrieve Cassette

  1. The microscope is not doing any operations.
  2. Column valves are closed.
  3. Temperature of all components is cryogenic.
  4. Position 1 (cross-grating) is loaded onto the stage or no grid is loaded.

  1. Krios: Push green dock Button (fig 6A).
  2. Arctica/Glacios: Push touchscreen “Dock” button.
  3. Any autoloader: Click “Load” on the microscope computer autoloader interface (fig 6B).

  1. Krios: “NanoCab locked” light off, “cassette in loader” light on (fig 6A).
  2. Arctica/Glacios: “NanoCab can be removed” displayed on the screen.
  3. Any autoloader: Interface says “You can now remove the NanoCab” (fig 6B).

4.4 If Cassette is Stored Warm: Cool Cassette

Avoid contamination: always wear gloves when handling anything that enters the microscope vacuum, including the cassette

  1. Using the cassette tweezers, securely grasp cassette at the top position (fig 7). [There are two indentations at the top of the cassette for the cassette tweezers. The Silver shield (foot) is the bottom of the cassette.]

4.5 Attach NanoCab to Cassette Loading Station and Positino Cassette for Loading

  1. Holding the cassette gripper handle, push the cold end into the NanoCab.
  2. Press and hold the side gripper button (see fig 8) to grip the cassette.
  3. In one fluid motion, being careful not to lift out of nitrogen vapor, remove the cassette from the NanoCab and place it in the loading position of the workstation (fig 8).
  4. Release the gripper button and slide the handle back to the home position. [The metal block has two support pins that fit the underside of the gripper (fig 10). Failure to place gripper on these pins will result in the end popping up out of the LN2 vapor and warming up.

4.6 Add Autogrids to the Cassette

  1. Re-cool the autogrid tweezers by briefly submerging in LN2.
  2. Add LN2 as required.
  3. Check for secure insertion in cassette by gently tapping the top edge of the autogrid with the autogrid tweezers

4.7 Add Loaded Cassette to the Attached NanoCab

4.8 Dock Nanocab with the Autoloader

  1. The microscope is not doing any operations.
  2. Column valves are closed.
  3. Temperature of all components is cryogenic
  4. Position 1 (cross grating) is loaded onto the stage (or no grid is loaded)

  1. Krios: Push green dock Button (fig 6).
  2. Arctica/Glacios: Push touchscreen “Dock” button.
  3. Any autoloader: Click “Load” on the microscope computer autoloader interface (fig 6B).

  1. Krios: “NanoCab locked” light off, “cassette in loader” light on.
  2. Arctica/Glacios: “NanoCab can be removed” displayed on the screen.
  3. Any autoloader: Interface says you can now remove the NanoCab (fig 6B, above)

4.9 Cleaning up

5) Additional Figures:

5.1 Figure 12. Autoloader Dock Highlighted

6) Chemicals:

    1. Liquid Nitrogen

7) Waste Disposal:

    1. Follow facility procedure for proper disposal.