Spray-freezing Rapid Freeze and Freeze Substitution of Unicellular Organisms for Transmission Electron Microscopy

A. Collection of material

  1. Obtain a dense culture of organism in an aqueous medium.
  2. Spray some of the organisms in culture medium at ambient temperature onto a microscope slide using an airbrush with compressed nitrogen gas. Adjust pressure and aperture size so that the cells are not damaged by spraying. The spray spots should be about 100 Ám in diameter.

B. Spray freezing

  1. Select specimen basket mesh that will be fine enough to hold the specimens and insert in spray freezing chamber.
  2. Cool spray-freezing chamber with liquid nitrogen until nucleate boiling is completed.
  3. Slowly add propane to spray-freezing compartment condensing liquid propane in the freezing compartment. Allow propane to reach about -188° C.
  4. Once propane has reached temperature, spray sample in short bursts into the specimen basket. Watch for the temperature to recover.
  5. Once spraying is completed, remove the basket quickly draining excess propane and then transfer the basket to pre-cooled acetone at -120°C.

C. Freeze substitution

  1. Material is dehydrated with two or three changes of acetone over two or three days at temperatures that are below -85° C. Cells are substituted at this temperature for 48 - 72 hours.
  2. Substitution tubes are then placed into holes drilled into a lead block at -85° C, transferred to a freezer and allowed to gradually warm to -20° C.
  3. Chemical fixation and staining may or may not be used. Here are two protocols:
    1. Anhydrous fixation: Material is fixed with anhydrous glutaraldehyde and/or osmium tetroxide in anhydrous acetone at -20°C. Fixation is for several hours in either fixative. (Mix and store all acetone-based osmium tetroxide solutions below -20°C or lower at all times.)
    2. Hydrous fixation: An alternative method to staining anhydrously is to rehydrate the material to water and then fix in 1% osmium tetroxide as in typical aqueous chemical fixation, with routine dehydration and infiltration following conventional protocols.
    3. Advantages & disadvantages: Anhydrous osmium tetroxide may not partition into lipids at cryogenic temperatures creating negative staining of membranes. Hydrous osmium tetroxide results in conventional images in which may of the advantages of cryofixation are retained, but more extraction occurs.

  4. Lead block transferred to refrigerator until it reaches 4° C.

D. Infiltration

  1. Infiltrate cells for 24 hours in each of the following: Spurr's resin : acetone, (1:2); Spurr's resin: acetone, (2:1); 100% Spurr's resin.
  2. Embed in fresh Spurr's resin and polymerize for 10 hours at 70° C.