Initial preparations

  1. Attach cryostage goniometer adaptor to the EM.
  2. Prepare silver dewar with black lid and spout with filtered liquid N2 and larger dewar for filling anticontaminators.
  3. 1200 holder
    1. Turn on HT (load AUTOHT; run; vstep 100 V, 1 sec).
    2. Align microscope (normally 120 kV for low dose) and set specimen position at 1.
    3. Insert Oxford blade anticontaminator into position, holding it against vacuum pressure. (Remove plastic stop and engage to the last slot).
    4. Fill microscope and specimen anticontaminators with liquid N2.
    5. Clean holder tip with velin (ethanol if dirty), grease O-ring with a tiny amount of fomblin grease.
  4. 2010 holder
    1. Fill microscope anticontaminator.
    2. Load and cool holder.

NB: on 2010 holder, never get grease or fingerprints on the yellow ring at the tip. Insert holder in microscope, remembering to slot the end of the bellows into the goniometer plate.

Cooling the holder dewar

  1. Use funnel of correct length only, to avoid scratching the smooth surface inside the dewar or flooding the indium seal in the neck with liquid N2.
  2. Connect lead to temperature control unit. Sensor position 1 is for holder temperature.
  3. Top up with two full funnels of liquid N2 allow to boil-off completely. DO NOT overfill - never leave liquid N2 sitting in the neck of the dewar.
  4. When the large plume of N2 ceases top up again with two more funnels full of liquid N2. Cap the dewar and observe a slow but consistent escape of gas. Monitor the decrease in temperature on the temperature controller. Check the level of liquid N2 in the dewar after a few minutes, top up again and use the tool to expel any liquid N2 in the neck of the dewar. It should cool down to -170° or better, within ~½ hr.

Cooling the workstation

When the sample and holder are ready:

  1. 1200 - make sure the workstation plunger is closed. 2010 - make sure the gate to workstation is closed. This avoids cooling the rod area of the workstation, which will affect the o-ring seal and the airlock pumping when the rod is inserted into microscope.
  2. Fill the silver dewar with liquid N2.
  3. Cool workstation with liquid N2, but avoid overfilling the bottom reservoir as this will overflow around the styrofoam insulation and cause frosting.
  4. 1200 - Fill up to aluminium plate. 2010 - Fill up to about 1cm below the rim of the styrofoam insualtion, through circular hole, subsequent fillings should top up through the rectangular troughs.

The workstation is ready when liquid N2 wets the aluminium plate and remains in the wells without boiling. Note: only introduce dry implements and filtered N2 into specimen chamber and holder dewar. Cold implements need to be kept inside the workstation to avoid frosting in air.


As soon as the workstation is cold, transfer holder with bellows extended from the microscope into the workstation.

  1. Switch on N2 gas at cylinder. Flush the gas lines through the workstation for about 1min to dry it. (Adjust cylinder regulator to give max 5-10 psi on workstation regulator). NB for 1200 one cylinder feeds both the microscope and the workstation - adjust direction of flow at tap. For 2010 use the cylinder marked workstation.
  2. Block gas flow to workstation.
  3. Put the cooling coil in the silver dewar. If a blockage occurs in the cooling coils or gas lines removal of the coil from the dewar is sufficient to clear it.
  4. Connect gas lines to bellows and goniometer.
  5. 1200 - Slide back workstation plunger (OPEN). 2010 - Open gate ot workstation.
  6. Open a small gas flow to the workstation about 5 psi.
  7. Splash the tip of the holder to cool it to about -190° then regulat the gas flow to 1 psi for loading the grid.
  8. Wear face mask to avoid breathing on specimen. Put the grid into the workstation. Cool locking ring, forceps and loading tool, and load grid container into workstation. Load the grid into the holder.
  9. Close the specimen shutter. Splash the tip and cool to -190°.
  10. Connect gas lines to bellows and goniometer.
  11. Close gas flow to workstation.
  12. Transfer holder to microscope. Allow the N2 to spill out freely when inserting the holder. (Follow the set procedures for inserting the holder into the 1200 and 2010). Specimen temperature should not go warmer than -160° during the transfer.
  13. Disconnect gas lines and switch off flow.

Re-filling the dewar

  1. The holder dewar should still be cold. Refill the dewar with two full funnels of liquid N2 lifting funnel to allow it to empty.
  2. Insert the flushing tool for a few secs (or until the fountain of N2 ceases to flow from the dewar) to blow off the excess and stop boiling.
  3. Hook up tubing lines and disconnect lead to the heater to prevent vibration.
  4. Wait for temperature to stabilize and frost to disperse before opening shutter and looking at grid (~15 min). Check for shorting by pressing button next to red light on anticontaminator (light should not get brighter), caused by frost between specimen and anticontaminator.
  5. Empty workstation, warm up and dry container and tools. Top up holder once every 2 hrs; anticontaminators once every 2-3 hrs. (2010 anticontaminator should last all day if fully filled).

Finishing procedure

  1. Cryo holder - slide tip of holder into metal sleeve, pour out N2 and pump the sleeve for at least ½ hr on rotary pump.
  2. Microscope
    1. 1200 holder - filament & HT off. Empty Oxford anticontaminator. Switch N2 gas cylinder to microscope flush line, flow rate 0.5 psi, to change plates. Once new films are loaded and camera is pumped down, insert heating coil into microscope anticontaminator, plug in socket and press ACD Heat button. This will warm up the anticontaminator and automatically shut off after 1-1½ hrs. If not, press the button after 2 hr or next day. Replace the goniometer adaptor.
    2. 2010 holder - ramp down filament current using program. Remove holder and protect tip with sleeve and pump as above. Switch on N2 gas to camera and change plates. When plates have pumped down, plug in socket and press ACD Heat button. Replace goniometer adapter.

Short instruction list for cryo transfer on 1200 EX

Prepare microscope and cryo holder

  1. Change goniometer adaptor (1200). Holder sample position 1.
  2. Fill dewars (silver one with black top, filtered)
  3. Insert Oxford anticontaminator to last position; fill built-in, then Oxford anticontaminator
  4. Insert cryo stage (clip bellows into black slot), fill dewar; connect temp control lead & power on
  5. Top up holder 2-3 times - takes ~½ hr to cool down to -160°. Don't overfill.
  6. Set up workstation and gas flow (max 10 psi)

When sample is ready

  1. Start gas flow. Fill cooling coil dewar (close plunger in workstation chamber)
  2. Fill workstation. Connect bellows and goniometer gas lines and transfer holder into workstation. Disconnect gas lines and reduce gas flow.
  3. Insert can with sample. Wear face mask
  4. Cool tools. Remove old grid and insert new sample (Adjust gas flow and make sure tip temperature is <-160°!!)
  5. Flush tip with liquid N2 and close specimen shutter
  6. Connect bellows and goniometer gas lines, pressure to 10 psi. Tip temperature to -190°.
  7. Transfer holder into microscope. Top of holder facing you, to get pin in correct orientation to activate airlock pumping. Wait 2 pump cycles.

Finishing cryo session

  1. Remove cryo holder from microscope and slide tip into the protective metal sleeve. Pour out N2 and pump out sleeve with rotary pump for ½ hr - overnight.
  2. Replace films, remembering to switch gas flush line, 0.5 psi
  3. Filament and HT off. Empty Oxford anticontaminator dewar and pull the anticontaminator to the out position. When camera chamber pumped down, insert heater rod into microscope anticontaminator dewar and plug into socket. Press ACD heat button. Should switch off automatically within ~1½ hr. If not, press button after 2 hr.
  4. Change goniometer adaptor