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    SEM preparationSEM preparation

    After dehydration withethanol, samples are

    Critical Point Dried

    (CPD)

    PurposePurpose: To completely dry

    specimen for mounting while

    maintaining morphological

    details.

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    If the temperature of liquefied gas is increased the meniscus becomes

    flatter indicating a reduction in the surface tension. If the surface tension

    becomes very small the liquid surface becomes very unsteady and

    ultimately disappears.

    When this 'critical point' is

    reached, it is possible to

    pass from liquid to gas

    without any abrupt change

    in state. If a specimen hadbeen in the liquid it would

    have experienced a

    transition to a 'dry' gas

    environment without being in

    contact with a surface,

    avoiding the possibility of

    the damaging effects of

    surface tension.

    This is termed Critical Point Drying (C.P.D.) the basis of

    which are the classic experiments carried out over 100

    years ago during investigations on the liquefaction of

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    Initial investigations were CO2 as will be apparent from

    Figure 2 - table of Critical Constants for some common

    substances. The critical conditions of other substanceswould not help biological material, as the specimens would

    suffer significant thermal damage if attempted.

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    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.

    MethodMethod

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    Sample holdersSample holders

    -Keep samples separated

    -Hold delicate or small

    samples

    -Ease of sample retrieval

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    Freeze Drying

    -Sample is quick frozen in

    liquid nitrogen (LN2).

    -Placed in vacuum evaporator

    on frozen block

    (approx. -190 C).

    -Left under vacuum for several

    days to sublimate water.

    -Mounted and coated.

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    HexamethyldisilizaneHexamethyldisilizane

    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

    Stain Technology, 1983, Williams & Wilkins vol. 5, NO. 6, p.347

    Biotechnic and Histochemistry, 1994, Williams & Wilkens vol. 69, no.4,

    p192

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    Mounting the specimen onto stubsMounting 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.

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    Conductivity of Samples

    Charging results in:deflection of the beam

    deflection of some secondary electrons

    periodic bursts of secondary electrons

    increased emission of secondary electronsfrom crevices

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    Coating the Sample

    -Using OsO4 as fixative (biological)

    -Painting a grounding line with silver or carbon paste

    -Coating with nonreactive metal or carbon

    a) Increased conductivity

    b) Reduction of thermal damage

    c) Increased secondary and backscattered electron emission

    d) Increased mechanical stability

    Accomplished by:Accomplished by:

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    Gold, gold palladium target

    -vacuum of approx. 2 millibar

    -thickness 7.5 nm to 30nm

    Sputter coatingSputter coating

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    Thermal evaporationThermal evaporation

    -Typically used for shadowing

    - 2 x10-7 torr-From coarse to fine:

    Carbon, gold, chromium,

    platinum, tungsten, tantalum

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    Evaporation

    Trough for powders/cleaning

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    E-beamUsed for high melting point

    metals (e.g. tantalum)

    Similar to create emission of

    electrons from filament in

    microscope

    Provides highest resolution

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    Carbon Coating

    Good vacuum required

    Carbon rod may need outgassing

    Do not look directly at heated electrodes

    For samples in SEM where

    x-ray information is needed.

    TEM grids needing extra

    support

    Support for replicas

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    Rotary device to

    ensure uniform

    coating

    Carbon ribbon


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