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)
- Glycerol, Sucrose, Ethylene glycol, DMSO
- Freeze sample
Liquid nitrogen (-196°C)
Freon 12 or 22 (-165°C)
- Freezing method
- Double replica vs. cup
- -100°C at 10-5 Torr or better
- Brings out relief of membranes
- -100°C to -120°C
- 0 to 60 sec. (~23Å/sec. at -100 °C)
- Resistance heating (or electron beam) evaporation
- Platinum 20Å, Carbon 200Å
- Thickness measured with quartz crystal
- 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