Cryo- Cryo- Preservation Preservation Rapid arrest of cellular components Rapid arrest of cellular components Avoidance of artifacts from chemical fixation Avoidance of artifacts from chemical fixation Preserves sample in hydrated state Preserves sample in hydrated state Maintains structural and cellular integrity Maintains structural and cellular integrity Cellular domains are maintained (e.g. IMPs) Cellular domains are maintained (e.g. IMPs) Ice crystal formation can be avoided Ice crystal formation can be avoided Sublimation (“etching”) used to remove excess water Sublimation (“etching”) used to remove excess water Structures are ephemeral or events are rapid. Structures are fixative sensitive. Removal of water changes topography/morphology Why?
28
Embed
Cryo-Preservation Rapid arrest of cellular components Avoidance of artifacts from chemical fixation Preserves sample in hydrated state Maintains structural.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Cryo-PreservationCryo-Preservation
Rapid arrest of cellular componentsRapid arrest of cellular componentsAvoidance of artifacts from chemical fixationAvoidance of artifacts from chemical fixationPreserves sample in hydrated statePreserves sample in hydrated stateMaintains structural and cellular integrityMaintains structural and cellular integrityCellular domains are maintained (e.g. IMPs)Cellular domains are maintained (e.g. IMPs)Ice crystal formation can be avoidedIce crystal formation can be avoidedSublimation (“etching”) used to remove excess waterSublimation (“etching”) used to remove excess water
Structures are ephemeral or events are rapid. Structures are fixative sensitive. Removal of water changes topography/morphology
Why?
DisadvantagesDisadvantages
Specialized equipment required
Freeze Damage
Limited view of specimen or difficulty manipulating frozen material
Slam FreezeSlam Freeze““Gentleman Jim”Gentleman Jim”
Liquid nitrogen container
Sample holder
High pressureHigh pressurefreezerfreezer
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.
TEM Plunge Freeze/ Freeze SubstitutionTEM 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
Automated temperature controllerAutomated temperature controllerfor freeze substitutionfor freeze substitution
EndoplasmicEndoplasmicreticulumreticulum
Fungus hyphal tipFungus hyphal tip
Cryo TEMCryo TEM
Image capture with frozen sectionsImage capture with frozen sections(Low dose monitored)(Low dose monitored)
Freeze-fractureFreeze-fracture
Sample is rapidly frozen, fractured and a replica is made.
Etching (sublimation) of the sample may be used to expose features.
Sample PreparationSample Preparation
Glycerol added if feasible.
Sample is put into holders (“hats” or planchets)
Fracture surface with frozen bladeFracture surface with frozen blade
Coat exposed surface with metal (30-45 o angle)
Reinforce by coating with carbon (90 o angle)
Fracture Plane ViewsFracture Plane Views
Fracture Cut
Single surface viewSingle surface view Two surface viewTwo surface view(both halves recovered)(both halves recovered)
TEM of Golgi sections TEM of Golgi sections and freeze fractureand freeze fracture
Freeze-fractured replica of Freeze-fractured replica of the alga Dunaniellathe alga Dunaniella
Conventional SEM Sample PrepConventional SEM Sample Prep
1. Aldehyde/Osmium2. Dehydration in solvent3. Critical point drying4. Mounting and Coating
Disadvantages:Disadvantages:Time of preparationTime of preparationNot all components are preserved Not all components are preserved
(e.g. carbohydrates, water)(e.g. carbohydrates, water)Possible collapse of structuresPossible collapse of structuresDamage during mountingDamage during mounting
SEMSEMPlunge Freezing and CryostagePlunge Freezing and Cryostage
Specimen holder and Specimen holder and transfer rodtransfer rod
Nitrogen slushing and Nitrogen slushing and plunge stationplunge station
Ice crystalIce crystalformationformation
Leidenfrost effectLeidenfrost effect
Cryofixed Yogurt - no fracture, 3hr etch
Cryofixed Fetafractured and 5 minute etch
Effects of EtchingEffects of Etching
Correlation - Light Micrographs and SEMCorrelation - Light Micrographs and SEM