Cryo-fixation is a technique used to immobilize the specimen at cryogenic temperatures. There are two techniques available in the BIF, plunge freezing and High Pressure Freezing (HPF).
Specimens are physically immobilized within ultra-thin vitrified (amorphous) ice layers. This technique is mainly used for freezing aqueous suspensions, such as macromolecules and cell suspension cultures.
B. High Pressure Freezing
Specimens are physically immobilized within milliseconds at high pressure and cryogenic temperatures. The sample must fit into a 3 mm or 6 mm diameter carrier. This technique is mainly used to preserve tissues that are 100 – 600 um thick.
Leica HPM100 High Pressure Freezer
Complete cessation of all cellular processes (non-selective, within msec)
Problems caused by osmotic shock or inappropriate pH, often associated with
chemical fixation, are not present.
Formation of ice crystals could damage the ultrastructure.
Comparison of High-pressure-freezing vs Chemical fixation:
|High Pressure Freezer||Chemical Fixation|
|Fixation Time||Ultra rapid (msec)||Slow, simply by diffusion|
|Fixation Efficiency||High, non-selective||
(A time lag in fixation between outer and inner tissue)
|Fixation Artifacts||Formation of ice crystal||
Many on TEM level
(Shrinkage, membrane distortion, aggregation of proteins/lipids)
|Sample Size||Must fit into sample carrier||Variable|