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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possi

Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possible. Avoid artifacts (changes, loss or additional information) Render specimen stable for examination in environment of SEM. Standard Preparation. Tissue. TEM. SEM.

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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possi

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  1. Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possible. Avoid artifacts (changes, loss or additional information) Render specimen stable for examination in environment of SEM.

  2. Standard Preparation Tissue TEM SEM Chem. Fixation Cryo Fixation Chem. Fixation Cryo Fixation Rinse/store Substitution Rinse/store En bloc staining Cryo- sectioning Dehydration Dehydration Dehydration Drying Resin infiltration Mounting Sectioning Coating Post staining

  3. SEM preparation After dehydration with ethanol, samples are Critical Point Dried (CPD)

  4. Critical point drying (CPD) Purpose: To completely dry specimen for mounting while maintaining morphological details.

  5. Method 1) Water exchanged for ethanol. 2) Ethanol exchanged for liquid CO2 (transitional fluid). 3) CO2 brought to critical point (31.1 C and 1,073 psi), becomes dense vapor phase. 4) Gaseous CO2 vented slowly to avoid condensation. 5) Dry sample ready for mounting.

  6. Sample holders -Keep samples separated -Hold delicate or small samples -Ease of sample retrieval

  7. Freeze Drying -Sample is quick frozen in liquid nitrogen (LN2). -Placed in vacuum evaporator on cold block (approx. -190 C). -Left under vacuum for several days to sublimate water. -Mounted and coated.

  8. Hexamethyldisilizane HMDS is a chemical method of “drying” the sample Primarily used with insects, larger fleshy tissues, soft invertebrates, etc... HMDS is a strong irritant and volitile (flammable). Brief protocol: -After fixation - ethanol dehydration to 100% -Transition from ethanol to HMDS -Two changes of pure HMDS -Left overnight in dessicator with silica gel

  9. Mounting the specimen onto stubs “Stubs” are specimen holders specific for the instrument being used (e.g. Zeiss or Hitachi SEM) Specimen is held to stub by conductive tape, paste or glue.

  10. Conductivity of Samples Charging results in: deflection of the beam deflection of some secondary electrons periodic bursts of secondary electrons increased emission of secondary electrons from crevices

  11. Coating the Sample a) Increased conductivity b) Reduction of thermal damage c) Increased secondary and backscattered electron emission d) Increased mechanical stability Accomplished by: -Using OsO4 as fixative (biological) -Painting a “grounding line” with silver or carbon paste -Coating with nonreactive metal or carbon

  12. Sputter coating Gold, gold palladium target -vacuum of approx. 2 millibar -thickness 7.5 nm to 30nm

  13. Thermal evaporation -Typically used for shadowing - 2 x10-7 torr -From coarse to fine: Carbon, gold, chromium, platinum, tungsten, tantalum

  14. Evaporation Trough for powders/cleaning

  15. Used for high melting point metals (e.g. tantalum) Similar to create emission of electrons from filament in microscope Provides highest resolution E-beam

  16. Carbon Coating For samples in SEM where x-ray information is needed. TEM grids needing extra support Support for replicas Good vacuum required Carbon rod may need outgassing Do not look directly at heated electrodes

  17. Carbon ribbon Rotary device to ensure uniform coating Carbon Rods

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