Acetone evaporates the film is placed on the still wet surface from one end so that there is no bubble formation. Allow surface tension forces to pull the film down against the specimen; no pressure is required and allowed to dry. As an alternative one surface of the replication film may be softened with solvent and then laid on the specimen surface. Make sure that no air bubbles are trapped during this procedure. (see note 1)
Stage 2: Steps for Making Positive Shadow Casting Replicas
The technique of shadow-casting consists of depositing by vacuum evaporation a layer of electron-dense material on the specimen at an angle. Due to shadowing some areas are more electron transparent thanothers giving the impression of illumination by oblique light when viewed by a TEM
- First stage replica is taped to a glass microscope slide, structure side up, with scotch tape.
- This is then placed into a vacuum evaporator and shadow cast with the desired material (see note 2) at the desired low angle and direction. It is then reinforced by coating with Carbon at an angle of 90 degrees to the surface. Some prefer to perform the Carbon coating first followed by the shadow layer.
- Prepare washing device by placing filter paper or wire mesh in Petri dish and completely wetting with solvent, then cover.
- Cut 2 – 3mm squares from shadowed and coated replica with a razor blade or scalpel. Digest the Cellulose Acetate negative by carefully placing these, acetate side down, on TEM grids and then place the grids on the wetted filter paper or wire mesh in the Petri dish, then cover. Add solvent as necessary to keep wet. Move grids several times to a clean area of paper. Acetate will be completely removed in less than an hour.
- Remove grids from dish and allow to dry before placing in TEM for examination.
- If the surface to be replicated is contaminated with foreign substances or to insure absolutely clean replicas, the surface should be cleaned by making several blank replicas to remove this contamination. This is especially true on etched surfaces of metal where corrosive materials are often found. 3-5 of these discarded replicas may be necessary.
- The choice of shadowing material is made according to the magnification to be used when viewing in the electron microscope. If the magnification is less than 3000x, Chromium, Palladium, or Germanium is used. When more than 3000x Platinum-Palladium alloy, Platinum, Iridium or Platinum-Iridium is used.
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