Introduction
The main idea of high-pressure freezing is to fix biological samples without the unpredictable effects of chemical fixation a life-like ultrastructural analysis.
Cryo-immobilization achieves best preservation of biological samples at the ultrastructural level, provided that the biological material is not distorted by the formation and growth of ice crystals. At ambient pressure, this requires high cooling rates (> 10’000 K/ s for a few µm) or antifreezing agents at low freezing rates. Specimens of up to 10 µm in thickness can be frozen without detectable ice crystal damage and without the use of intracellular antifreezing agents by liquid propane jet freezing. Thicker samples can be adequately frozen only when the physical properties of water are changed, e.g., using antifreezing agents or under high pressure (210 MPa). The thickness of biological specimens to be adequately frozen by high pressure freezing is limited to about 200 µm.
The formation of ice crystals can be greatly reduced if the specimen is subjected to a pressure of 2100 bar before the freezing process has started. Pressure may cause changes in the physical and chemical equilibrium, such as dissociation or the reactivity of enzymes. This must be prevented in order to immobilize the specimen in a near native state. In the high-pressure freezer, a pressure of 2100 bar is applied within a few milliseconds, the specimen immediately frozen with pressurized liquid nitrogen and transferred automatically to liquid nitrogen at ambient pressure (-196°C).
Tools
Parts
No parts specified.
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You will find the High-pressure freezer Leica EM HPM100 in Y42H86.
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Close all valves at the back of the machine.
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Taps are kept open after air heating to let condensation water escape.
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Closed position of the valves.
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Switch on the system at the main switch on the front panel.
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Connect the Leica USB stick to the HPF to save the process parameters for every freezing cycle (if not plugged in already).
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On the display of the HPF enter the current date as the folder name (YYYYMMDD).
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To turn off the alcohol select "SYSTEM" from the main screen.
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and press "ALKOHOL ON".
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The display button changes to "ALKOHOL OFF".
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The machine is ready to freeze without alcohol injection, which is indicated on the main screen.
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To add 100% isopropanol or ethanol, slide the binocular to the right until it comes to a stop.
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Open the front safety cover and prop it using the cover support lock.
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Unscrew the alcohol tank cap and fill the tank with 40 mL isopropanol or ethanol.
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After filling, close with the tank cap (hand tight only). Close the safety cover by pushing the cover support back to release the lock.
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Add liquid nitrogen to the storage dewar, until the front display shows at least 70%. If many samples will be processed fill 100%. A small pop-up rod in the top cover rises when the dewar is completely full of liquid nitrogen.
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Fill the specimen dewar before freezing only halfway with liquid nitrogen to avoid the samples from jumping out due to boiling nitrogen.
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The system needs to build up the pressure prior to freezing.
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The system is now "ready for freezing"
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Pull machine to front to get access to tubing on the back side. Put big styrofaom box below tubes.
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Open valves at the back to release nitrogen into styrofoam box.
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Once the storage dewar has reached a level of below 20% select "SYSTEM" from the main screen.
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Select "AIR HEATER ON".
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This activates the heating cycle. Cycle will only start when the chamber temperature reaches 10°C.
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