In the freeze fracturing process, a specimen is frozen rapidly and cracked on a plane through the tissue. This fracture occurs along weak portions of the tissue such as membranes or surfaces of organelles.

After cleaving, both surfaces are shadowed with a platinum film. This coating produces a replica of the surfaces. The replica is then coated with carbon and is then imaged in the transmission electron microscope.

While there are several uses for freeze fracture, one of the mostcommon is to determine the prescence of tight or occluding junctions where membrane glycoproteins bind cells together. Freeze fracture is the only way to determine the prescence of such junctions.

The method is also good for the study of intramembrane structures.

Sample Preparation for Freeze Fracture

Fix sample (typically glutaraldehyde)

Cryoprotect
Glycerol, Sucrose, Ethylene glycol, DMSO

Freeze sample
Cryogens
Propane (-187°C)
Liquid nitrogen (-196 °C)
Freon 12 or 22 (-165 °C)
Freezing method
Dip
Cryojet
Spray

Fracturing
Double replica vs. cup
-100 °C at 10-5 Torr or better

Etching
Brings out relief of membranes
-100 °C to -120 °C
0 to 60 sec. (~23Å/sec. at -100 °C

Replication
Resistance heating (or electron beam) evaporation
Platinum 20Å, Carbon 200Å
Thickness measured with quartz crystal

Digestion
Warm specimen to room temperature
Sodium hypochlorite (bleach)
Nitric, sulfuric, and chromic acids
1 to 4 hours

Pick up replica with TEM grid and image in microscope