CRYO-SAMPLE PREPARATION

Cryo-processing

Cryo-immobilization 

• Bal-tec High Pressure Freezer. The Balzers HPM 010 high pressure freezer is available for rapid, cryo-immobilization of samples. Specimens are subjected to liquid nitrogen and a pressure of 2000 bar in a few milliseconds. The water of the sample is vitrified under this regime, retarding ice-crystal formation, thereby avoiding structural damage to the specimen associated with ice-crystals. The pressure immediately prior to and during the freeze results in a very viscous water, reducing ice nucleation (Water Phase Diagram). The frozen specimen may then be fixed and the water substituted while in the solid phase, or minimally etched, coated and viewed in the SEM equipped with a cryo-stage. Alternatively, frozen samples may be freeze-dried and critical point dried for viewing with TEM or SEM at ambient temperature. The maximum sample size to fit into the high pressure freezing planchette is 1mm in diameter; vitrious ice can be achieved in samples up to several hundred microns. High pressure freezing lowers the freezing point of water, enabling large samples to be frozen without obvious ice nucleation.
  
• Plunge freezer. Plunge freezing can result in good ultrastructural preservation in samples up to 15 microns. Routinley samples are plunge frozen on EM grids.

   

Freeze-fracture, freeze-etching/drying and metal coating play an important role in biological specimen preparation and cryo-preparation following cryo-immobilization. High quality imaging of biological structures can be obtained when combining advanced thin metal film coating techniques with high resolution SEM.

• Bal-Tec BAF 400K Freeze-etching System. The Bal-Tec BAF 400K Freeze-etching system is equipped with a cryo-pumping system, 2 electron-beam guns, a quartz crystal thickness monitor, a gas partial pressure analysis, and an airlock system. It is configured to optimize for a clean chamber vacuum and coating at low temperature. This system is used for freeze-fracturing, freeze-etching/drying, double-layered coating of Pt/C and C on frozen-hydrated specimens (to reduce irradiation damage), double-axis rotary shadowing for high resolution cryo-SEM, and unidirectory or rotary shadowing.