Principles & Protocols

Principles - High Pressure Freezing

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Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Liquid nitrogen cannot normally be used for freezing because of the Leidenfrost effect; the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. Increasing the pressure raises the vitreous freezing temperature. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen hydrated state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Pressure wave cracks samples

Filler selection - Looking for an osmotically inactive hydrophilic substance

Buffers are bad - salts crystalize

Penetrating Fillers

Glycerol
Ethylene glycol

Non Penetrating Fillers

1-Hexadecene
- Vacuum infiltrate samples with hexadecane
- Excess hexadecane not a problem will blow off in HPF
BSA (10-20%)
Polyvinylpyrrolidone (PVP) (15%)
Dextran (15-25%)
Ficoll (5-15%)
low-melt agarose (0.5-2%)
Cold water fish gelatin (50-100%)
Yeast comes from Drosophila food.
Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Hat selection

Gold hats used for replicas because they will not dissolve in HCl
Water sticks to copper hats
Use stainless steal spacers between hats To mimic thickness of hat To use with sapphire disks to substitute hats

Solvent selection

Isopropanol is needed for freeze fracture only
Ethanol (technical) is okay for HPF

Loading

Use 1-2% agar as work surface. Prevents drying
Work fast
Don't let cells dry out
Use right filler
Use smallest volume
Spacer grids
- Slot grid or "Chein" grids (EMS or Pella):
- 27 um thickness or
- rhodium maxteform spacer, silver 50 um thickness
hat on top is dipped in hexadecene or set it on filter paper saturated with hexadecene in Petri dish.
Max dimension for specimen between hats is 200 um without cryoprotectants
Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
Hexadecene is a good lubricant to keep hats from sticking to the holder.

Embedding

en face.
- Could use flat molds and place hats at the tip and try to cut in cross section.
- reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
sapphire disk tricks
- Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
- Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
- Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
- 72hrs at -90degC followed by overnight at either -60 or -30 deg C
- temp gradients at 5deg/hr
Other variations:
- 5 hr at -90 was ok for recent studies of fly eggs
- -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

3 washes with acetone, ~10 min each

Protocol for yeast

Log-phase growth (OD 0.3-0.5)
Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
Place filter on wet filter paper or agar
Make spatula out of toothpick by cutting across the tip
Scrape up a bit of yeast from the filter and place in the hat (100 um well)
Cover with hexadecene-coated flat
Yeast stays in the 100um well after freezing
During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

-- BillRice - 10 Aug 2009