1. Cryo-Preservation Rapid arrest of cellular components Avoidance of artifacts from chemical fixation Preserves sample in hydrated state Maintains structural and cellular integrity Cellular domains are maintained (e.g. IMPs) Ice crystal formation can be avoided Sublimation (“etching”) used to remove excess water W Structures are ephemeral or events are rapid. W Structures are fixative sensitive. W Removal of water changes topography/morphology Why?

2. Disadvantages Specialized equipment required Freeze Damage Limited view of specimen or difficulty manipulating frozen material Hazards of using some cryogens

3. CryogensMelting ptBoiling pt Freon 13-181- 81 Isopentane-16028 Propane-189-42 Nitrogen-209-196 Ethane-183-88 Helium-272 (1 o K)-269

4. Equipment for Freezing DeviceFreezing depth ( cost DeviceFreezing depth (  m) cost Plunge freezer10-20.50 -50 Spray freezer10-2010-50 Slam freezer20-402K Propane Jet40 10K High Pressure50-100150K

5. Cryo Sample Preparation For TEM thin section Sample / holder are rapidly plunged into liquid propane kept at liquid nitrogen temperatures Propane / Freon / Ethanol LiquidNitrogen (-196 c)

6. Slam Freeze “Gentleman Jim” Liquid nitrogen container Sample holder

7. High pressure freezer Sample is placed in small hats with a cryogen (antifreeze) Placed in chamber and rapidly jet sprayed with ultra cold ethanol while simultaneously brought to high pressure. Sample then transferred to substitution fluid or cryoultramicrotomed.

8. TEM Plunge Freeze/ Freeze Substitution Frozen sample transferred to -80 o C substitution fluid (acetone or methanol, 4%OsO 4 ) Kept at -80 for 2 days, then gradual transition to warmer temps Acetone/methanol washes to remove OsO 4 Infiltration and embedding

9. Automated temperature controller for freeze substitution

10. Endoplasmicreticulum Fungus hyphal tip

11. Cryo TEM

12. Image capture with frozen sections (Low dose monitored)

13. Freeze-fracture Sample is rapidly frozen, fractured and a replica is made. Etching (sublimation) of the sample may be used to expose features.

14. Sample Preparation Glycerol added if feasible. Sample is put into holders (“hats” or planchets)

15. Fracture surface with frozen blade

16. Coat exposed surface with metal (30-45 o angle) Reinforce by coating with carbon (90 o angle)

17. Fracture Plane Views Fracture Cut

19. Single surface view Two surface view (both halves recovered)

21. TEM of Golgi sections and freeze fracture

22. Freeze-fractured replica of the alga Dunaniella

23. Conventional SEM Sample Prep 1. Aldehyde/Osmium 2. Dehydration in solvent 3. Critical point drying 4. Mounting and Coating Disadvantages: Time of preparation Not all components are preserved (e.g. carbohydrates, water) Possible collapse of structures Damage during mounting

24. SEM Plunge Freezing and Cryostage Specimen holder and transfer rod Nitrogen slushing and plunge station

25. Ice crystal formation Leidenfrost effect

26. Cryofixed Yogurt - no fracture, 3hr etch Cryofixed Feta fractured and 5 minute etch Effects of Etching

27. Correlation - Light Micrographs and SEM Cryo-fixed Whole Peanut Peanut Butter P CW S

28. Uncooked Rice Cooked