626 cryo-holder training video – transcript

• (0:00) Getting started

• To get started release the cryo holder from the pumping station

• Always close the valve on the holder body first

• Close the valve on the side of the pump by turning righty-tighty

• Close V1 and V2. When these are horizontal they’re closed

• This video covers the use and care of the Gatan 626 cryo-holder, used for routine imaging. It does not apply to other holders. Cryo holders cost around forty thousand pounds and they are fragile, so it is important to treat them with respect.

• (0:29) Getting started and safety

• Remove the cable and tubing from the holder body and turn the holder upright

• Never store the holder outside of one of the 3 safe places: the microscope, the pumping station or one of the transfer stations

• Holding the pumping station with your other hand, pull the holder back carefully. As you're pulling against a vacuum it will release quite suddenly. Carefully take it to the microscope

• (0:55) To the microscope

• Put the holder in the microscope as usual, flick the switch and wait for the green light to appear

• after the vacuum pump has caught you can walk away. This is a good moment to go and fetch nitrogen and top up the ACD

• Wear eye protection and a lab coat when handling liquid nitrogen. A cryo-apron is available if you wish to use it.

• (1:35) To the microscope continued

• When the green light comes on, further insert the holder and fill the dewar with liquid nitrogen. It will take about 15 minutes to cool the holder down enough to be used

• Before using the holder, pre-cool a transfer station. Use the plug and the lid to make sure it doesn’t ice up. Give it some time to cool down and allow the bubbling to subside. Do not fill it above the top of the aluminium insert

• (1:58) Removing holder from microscope

• Check Apertures, Beam, Centre stage and Detector as for other holders. Now remove the holder from the microscope. Note that nitrogen will spill out of the holder as you turn it, catch this with a jug in your other hand

• Flip the switch and wait for the green light to go off. Take the plug out of the transfer station, take the holder out and slide it into the station

• Top up the nitrogen with a little bit of the nitrogen that you just caught

• Open the frost shield, pre-cool the clip ring tool and if you haven’t done so already make sure that your grid box is in the appropriate slot

• (2:48) Exchanging grids

• To exchange grids, fill the transfer station with liquid nitrogen to the top of the aluminium insert of the transfer station.

• Make sure your grid box is in the slot and open it using a screwdriver.

• Pre-cool the end of the clip ring tool that has the silver screw thread on

• Wait until it stops bubbling and then turning right screw the clip ring onto the tool and remove the clip ring from the holder take care not to cross thread the clip ring

• (3:19) Exchanging grids continued

• pre-cool you tweezers before you touch the grid otherwise it will defrost

• catch your grid - in this video that takes me a good 20 seconds so you know it's not always easy

• And place it in the holder try not to bend or otherwise mangle the grid if you can - there we go - and

• (3:58) Exchanging grids further continued

• Click the clip ring back into the slot and unscrew the tool from the ring anticlockwise or lefty loosey

• cool the other side, the flat black side, of the clip ring tool and just give a little push on the clip bring to make sure that it seated properly

• top off the nitrogen and close the frost shield

• (4:27) Back to the microscope

• Take the holder back to the microscope and insert it

• as we haven't topped up the dewar in between taking the holder out and putting it back in again, no new nitrogen comes out.

• Hold on to the dewar body until you hear the click from the vacuum pump to show that the vacuum has kicked in and you can let go

• (4:50) Waiting for the green light

• This is an excellent time to go and tidy away the cryo station and get some more nitrogen were waiting for the green light in real time this is also a good moment to save any images that were left open and to make sure that you put in the correct specimen name on the computer

• Don't wander off too far because you don't want the holder to warm up more than is necessary so as soon as a green light goes on you want to turn the holder and get it upright so that you can fill it with nitrogen again.

• After the top up it will take a little bit of time to get the vacuum down and to get everything to stop drifting

• There we go. Insert the holder and top up the nitrogen.

• (5:35) Finishing your session

• Once you’ve finished your session take the holder out of the microscope, tip out any remaining nitrogen and slide it back into the pumping station. When it’s fully in rotate it upside down and slowly open first the bottom valve and then the top one. Connect the tubing and the correct cable

• Don’t forget to shut down the microscope as usual!

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