Cryo-Fixation

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).

A. Plunge-Freezing

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.

Equipment:

Leica Vitrobot

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.

Equipment:

Leica HPM100 High Pressure Freezer

Advantages:

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.

Disadvantages:

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

Variable

(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