High-pressure freezing of cell cultures grown on sapphire disks

Prepare your tools and parts for high-pressure freezing. In the cupboard above the machine you will find:

the three-piece cartridge consiting of an upper cylinder, special middle plate 6 mm and lower cylinder

spacer rings 6 mm (200 μm)

carriers 0.1/0.2 mm

filter paper

mini-rack for middle plates

metal block

petri dishes

In the cupboard on the main floor you will find:

2x HPF-tweezers

1x orange tweezer

LN2 sample storage box

box with 1-hexadecene Eppendorf tubes

screwdriver for LN2 sample storage box

EtOH glas beaker

heating plate

Prepare all tools and accesories required for your cryofixation experiment.

Check if the 200 µm spacer rings are completely flat otherwise replace them.

Organize the required 100/200 cavities on a filter paper in a petri dish.

For 6 mm sapphire disks the 100 µm cavity must face the cells on the sapphire disk.

Insert the two-piece cartridge consisting of an upper cylinder and lower cylinder in the loading mechanism.

Overview of sandwich configuration

Visualization of sandwich configuration

Hold the sapphire at 45°.

Align the edge within the recess of the middle plate.

Release it.

Take a aluminium specimen type A (100/200 µm cavity) carrier with 100 µm cavity down and only wet this side with 1-hexadecane.

Add the 200 µm spacer ring on top.

Move the middle plate to the loading station.

Blot excess liquid away using filter paper.

Important here is that you blot fast and put some pressure to prevent shifting of the 200 µm spacer ring.

Flip the loading lever 180° until the process button is in position.

Initiate freezing by pressing the button.

After cryofixation, the two-piece cartridge and the specimen carriers are automatically ejected into the specimen dewar and are ready for transfer.

Open the sample dewar drawer and transfer the sample dewar to LN2 box.

Always pre-cool the tweezers prior to use.

Transfer all components including your sample to the LN2 box.

Always pre-cool the tweezers prior to use.

First, transfer the sapphire disk to the LN2 sample storage box.

Then transfer the specimen carriers to a beaker filled with 70% EtOH for cleaning.

Place the sample dewar back into the high-pressure freezer drawer.

Place specimen carriers to a hot plate for drying.

Once all freezing is complete the two valves at the back can be opened. Allow LN2 to drain.

Once the storage dewar has reached a level of below 20% select SYSTEM form the main screen.

Select AIR HEATER ON.

This activates the heating cycle. The 80°C heating cycle will only start once the chamber temperature warms up to 10°C.

The bake out will automatically terminate at the end of the cycle leaving the instrument ready for the next user.

NOTE: Leave the main power to allow the heating cycle to run.

Once the air heating cycle is started, the compressor turns on "air heater working".

Once the cycle is complete the machine switches back to the standard screen and you can turn it off using the switch at the front.