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Cryo-Preservation

Cryo-Preservation. Why?. Structures are ephemeral or events are rapid. Structures are fixative sensitive. Removal of water changes topography/morphology. Rapid arrest of cellular components Avoidance of artifacts from chemical fixation Preserves sample in hydrated state

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Cryo-Preservation

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  1. Cryo-Preservation Why? • Structures are ephemeral or events are rapid. • Structures are fixative sensitive. • Removal of water changes topography/morphology 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

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

  3. Cryogens Melting pt Boiling pt Freon 13 -181 - 81 Isopentane -160 28 Propane -189 -42 Nitrogen -209 -196 Ethane -183 -88 Helium -272 (1o K) -269

  4. Equipment for Freezing Device Freezing depth (m) cost Plunge freezer 10-20 .50 -50 Spray freezer 10-20 10-50 Slam freezer 20-40 2K Propane Jet 40 10K High Pressure 50-100 150K

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

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

  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 -80oC substitution fluid (acetone or methanol, 4%OsO4) Kept at -80 for 2 days, then gradual transition to warmer temps Acetone/methanol washes to remove OsO4 Infiltration and embedding

  9. Automated temperature controller for freeze substitution

  10. Endoplasmic reticulum 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

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

  19. TEM of Golgi sections and freeze fracture

  20. Freeze-fractured replica of the alga Dunaniella

  21. 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

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

  23. Leidenfrost effect Ice crystal formation

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

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

  26. Rice Uncooked Cooked

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