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

Hazards of using some cryogens

CryogensCryogens Melting ptMelting pt Boiling ptBoiling pt

Freon 13 -181 - 81

Isopentane -160 28

Propane -189 -42

Nitrogen -209 -196

Ethane -183 -88

Helium -272 (1o K) -269

Equipment for FreezingEquipment for Freezing

DeviceDevice Freezing depth (Freezing depth (m) costcost

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

Cryo Sample PreparationCryo Sample PreparationFor TEM thin sectionFor TEM thin section

Sample / holder are rapidly plunged into liquid propane kept at liquid nitrogen temperatures

Propane / Freon / Ethanol Propane / Freon / Ethanol

LiquidLiquidNitrogenNitrogen(-196 c)(-196 c)

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

Cryo-fixed Whole PeanutCryo-fixed Whole Peanut Peanut ButterPeanut Butter

P

CW

SS

UncookedUncooked

RiceRice

CookedCooked