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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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slide1

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

slide2

Disadvantages

Specialized equipment required

Freeze Damage

Limited view of specimen or difficulty manipulating frozen material

Hazards of using some cryogens

slide3

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

slide5

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

slide6

TEM Sample Preparation

Propane / Freon / Ethanol

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

Liquid

Nitrogen

(-196 c)

slide7

Slam Freeze

“Gentleman Jim”

slide9

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

slide10

Automated temperature controller

for freeze substitution

slide11

Endoplasmic

reticulum

Fungus hyphal tip

slide14

Freeze-fracture

Sample is rapidly frozen,

fractured and

a replica is made.

Etching (sublimation) of the sample may be used to expose features.

slide15

Sample Preparation

Glycerol added if feasible.

Sample is put into holders (“hats” or planchets)

slide17

Coat exposed surface with metal (30-45 o angle)

Reinforce by coating with carbon (90 o angle)

slide20

Two surface view

(both halves recovered)

Single surface view

slide23

Freeze-fractured replica of

the alga Dunaniella

slide24

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

slide25

SEM

Plunge Freezing and Cryostage

Nitrogen slushing and plunge station

Specimen holder and

transfer rod

slide26

Leidenfrost effect

Ice crystal

formation

slide27

Effects of Etching

Cryofixed Feta

fractured and

5 minute etch

Cryofixed Yogurt - no fracture, 3hr etch

slide28

Correlation - Light Micrographs and SEM

CW

S

P

Peanut Butter

Cryo-fixed Whole Peanut

slide29

Rice

Uncooked

Cooked