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Shadowing and Replicas Direct – TEM sample is coated and viewed

Shadowing and Replicas Direct – TEM sample is coated and viewed Indirect – Replica is made and viewed. One or two step method. SEM: Vinyl or plastic replica made by infusion of tissue, then original is removed. (Negative cast, corrosion casting)

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Shadowing and Replicas Direct – TEM sample is coated and viewed

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  1. Shadowing and Replicas Direct – TEM sample is coated and viewed Indirect – Replica is made and viewed. One or two step method. SEM: Vinyl or plastic replica made by infusion of tissue, then original is removed. (Negative cast, corrosion casting) TEM: Replica of surface is made (e.g. freeze fracture).

  2. Direct shadowing Negative Stained Bacteria Shadow cast of bacterium

  3. Materials for Shadow Casting Metal Melting point (C) Granularity Carbon 3650 Extremely Fine Chromium 1900 Fine Gold 1064 Coarse Platinum 1774 Fine Tungsten 3382 Extremely Fine

  4. Approximating Carbon Thickness Porcelain next to specimen, with drop of oil or bright white filter paper Light tan 5 nm Light chocolate 10 nm

  5. Shadow casting To enhance contrast and reveal topographic features by spraying particles of vaporized metal onto whole specimens

  6. Shadow Casting Vacuum evaporation of metal at shadow angle (a)

  7. Calculating Height of Shadowed Specimens H = height of specimen b = height to filament from specimen level c = distance from filament to specimen l = length of shadow H = b/c (l)

  8. Shadow Direction - Metal Source Angle Raised? Sunken?

  9. (Hall and Litt: Morphology of DNA macromolecules) Sumners, D. 1995. Lifting the curtain: Using topology to probe the hidden action of enzymes. Notices of the AMS 42:528-537.

  10. One Stage and Two Stage Replicas

  11. Corrosion casting • - The species of interest is anesthetized and anticoagulated 30 minutes before use. • - The abdominal cavity is opened and the aorta (or an artery supplying the organ of interest) is cannulated and the blood is flushed from the organ with saline or Ringer solution • - Resin (e.g Mercox/methyl-methacrylate/catalyst) is infused through the same cannula until the onset of polymerization (usually 10 minutes). • The resin-filled tissue is immersed in hot water (50° C) for one hour to complete resin curing. Tissue is removed by maceration in alternating rinses of 5% formic acid (15 minutes) and distilled water.

  12. Dried by lyophilization, and mounted on stubs for routine SEM. • Casts are sputter coated with gold and viewed at 10 kV in the SEM. • For vascular volume determination, resin filled tissues are weighted before and after masceration, and vascular volumes are calculated from tissue and resin densities. • Fixation of the tissue with dilute aldehyde fixatives prior to casting enhances preservation of details of the endothelial cells as imprints on the casts, but may prolong the maceration process

  13. Corrosion casting Fish gill Bullfrog eye - capillary Heart - capillary bed of trabicular muscles

  14. Nucleic Acid and Macromolecule Shadowing • 1. Nucleic acids suspended in hyperphase soln • 2. Solution allowed to flow down a CLEAN slide into a hypophase solution (either water or dilute salts) • Colloidion-coated grids used to pick up the nucleic acids floating on hypophase. Blot gently to remove excess liquid. • Grids floated on stain (e.g. Ethanolic UA), then washed in ethanol • 5. Once dry, grids are rotary shadowed and then viewed.

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